ABSTRACT
A STUDY OF WHEAT SEEDLING NUCLEASE AND ITS PROPERTIES

RELATING TO THE CATALYTIC MECHANISMS INVOLVED IN THE
HYDBOLYSIS OF DNA. RNA. AND THE 3'-NUCLEOSIDE MONOPHOSPHATES

By

Warren Dean Kroeker

Wheat seedling nuclease has been purified over 10.000
fold with a 5% yield of activity. This procedure produced 0.5
mg of protein from 7 kilograms of wheat. The isolated prep-
aration was Judged to be nearly homogenous in regards to size
and shape as indicated by the following results: a) sediment-
ation in the analytical ultracentrifuge revealed a single.
symmetrical boundary. b) sedimentation through a sucrose gra-
dient resulted in a single. coincident peak of protein and
enzyme activity. c) filtration in Sephadex 6-100 yielded a
single protein-activity peak. throughout which the activity
remained constant. and d) polyacrylamide gel electrophoresis
in the presence of 2-mercaptoethanol and SDB revealed the
presence of only one major protein band comprising greater
than 90-95$ of the total protein stained on the gel.

However. when electrophoresis of this preparation
of wheat seedling nuclease was performed at pH 8 in the
absence of SOS and 2-mercaptoethanol. 6-12 protein bands
were detected. Furthermore. the 3 activities associated
with wheat seedling nuclease were associated with only one
of these bands. The band responsible for the enzyme act-
ivities was one of the least negatively charged bands and
contained less than 10$ of the total protein stained on

Warren Dean Kroeker
the gel. All of the multiple-ionic forms of wheat seedling
nuclease were determined to be approximately equal in size.
This conclusion is based on the fact that when the percent
acrylamide contained in the gel was plotted as a logarith—
mic function of the relative mobility. similar slopes
were obtained for all of the bands.

The molecular weight of wheat seedling nuclease was
determined to be approximately h3.000 as determined by
the following methods: a) sedimentation velocity exper-
iments on the analytical ultracentrifuge. b) sucrose gra-
dient sedimentation with proteins of known molecular weights.
c) SDS gel electrophoresis with a system calibrated with
standard proteins. Since the SDS electrophoresis procedure
yielded a molecular weight which was similar to the other
two methods. it is likely that wheat seedling nuclease
exists as a single polypeptide chain.

Kinetic analysis of the rates of hydrolysis of 3'-AMP
and pApA by wheat seedling nuclease was performed by using
a continuous. spectrophotometric assay developed for this
purpose. The assay was based on the decrease in absorbance
produced when either adenosine or 5'-AHP (products of the
reaction catalyzed by wheat seedling nuclease) was deaminated
by a specific amino hydrolase coupling enzyme. The Km and
[Vmax values obtained for the dephosphorylation of 3'-AMP
were 1.5 x 10-5 M and 1.3 x 102;Lmoles/min/mg respectively.
The hydrolysis of pApA yielded Km and Vmax values of
9.1 x 10"5 w and 1.67 x 103,4lmoles/min/mg.

Kinetic inhibition studies were conducted using

warren Dean Kroeker
3'-AHP as the substrate. 5'-AMP. a molecule containing a
single phosphomonoester linkage. was found to be a com-
petitive inhibitor while the phosphodiester compounds.
2',3‘-cyclic AMP and ApA. were found to inhibit the reaction
in a noncompetitive manner.

A general salt inhibition was noted for wheat seed-
ling nuclease. The 3'-AMPase reaction was inhibited
competitively by Na01. However. when NaCl was included in
the pApA hydrolysis reactions. noncompetitive inhibition
resulted.

A consistent order of base preference for all three
of the activities associated with wheat seedling nuclease
was observed at pH 5.0. This preference was found to be
A7 ND) 7 C 7 G. Phosphodiester bonds adjacent to cytidylic
and guanylic acid residues were found to be quite resistant
to cleavage by wheat seedling nuclease.

When high molecular weight. native g, 22;; DNA or
intact. linear-duplex viral DNA molecules are hydrolyzed
by wheat seedling nuclease. only a few. specific double-
stranded cleavages occur. The resulting “limit polymers“.
still of high molecular weight. are resistant to further
hydrolysis by wheat seedling nuclease.

The nature of the sites on the duplex DNA recognized
by the enzyme was investigated. The production of limit
polymers (MW 2 3 x 106) from gh-l DNA (MU - 23 x 106) was
accompanied by the release of 0.0h-0.08$ acid-soluble
material. This corresponds to less than 8 nucleotide

equivalents released as acid-soluble material for each limit

Warren Dean Kroeker
polymer produced by the action of wheat seedling nuclease.
The 5' termini of limit polymers labeled with 32p by the
action of polynucleotide kinase contained either cytidylic
or guanylic acid. but no adenylic or thymidylic residues.

Wheat seedling nuclease exhibits a very large pre-
ference for denatured DNA as compared to native. double-
stranded DNA. However. the fact that wheat seedling nuclease
can hydrolyze the double-stranded. synthetic polymer. poly
d(A-T) at rates comparable to the hydrolysis of denatured
DNA. indicates that wheat seedling nuclease may recognize
A-T rich areas in native DNA. This contention is further
supported by the following evidences a)).DNA. which contains
an ApT rich region near its center. is rapidly cleaved to
produce A.halves. b) wheat seedling nuclease possesses a
considerable preference for bonds adjacent to A and T
residues while showing a marked resistance to the hydrolysis
of bonds adjacent to C and 6 residues. c) the nucleotide
composition of an area within 118 nucleotides of the 3' end
of the gh-l DNA limit polymers showed a slight increase in
APT content as compared to the whole gh-l DNA molecule and
d) the hydrolysis rate of native ).DNA increased 15 fold
between 20° and 30° C indicating a major structural effect

of the increased temperature on the substrate.

A STUDY OF WHEAT SEEDLING NUCLEASE AND ITS PROPERTIES
RELATING TO THE CATALYTIC MECHANISMS INVOLVED IN THE

HYDROLYSIS OF DNA, RNA AND THE 3'-NUCLEOSIDE MONOPHOSPHATES

BY

Warren Dean Kroeker

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Biochemistry

197”

Dedicated to

Barbara and Kristi

11

ACKNOWLEDGMENT

The author wishes to express his gratitude to Dr.
James L. Fairley for his guidance throughout the course
of this investigation. Discussions of both a scientific
and non-scientific nature contributed to a meaningful
experience as a graduate student. Also, thanks are
extended to Dr. William C. Deal, Dr. Fritz M. Rottman,
Dr. John C. Speck and Dr. Clifford J. Pollard who served
as his guidance committee. In addition, the author would
like to extend his appreciation to Dr. John A. Boezi and
Dr. Clarence H. Suelter for their time and assistance

during the course of this study.

111

INTRODUCTION . . .
EXPERIMENTAL PROCE
Materials ,
Resins
Protei
Enzyme
Nuclei
Bacter
Chemic
Wheat
Methods . .

Assays
Nuclea

TABLE OF CONTENTS

DLJRE O O O O O O O O O O O O O 0
ns 0 O C O O O O O O O O O O C O

S . . . . . . . . . . . e . . o
c Acids . . . . . . . . . . . .
ia and Bacteriophages . . . . .
als . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .

for Activity of Wheat Seedling

se 0 O O O O O O O O O O C O C 0

Standard Acid Soluble DNase and
RNaseAssaYaaaeaaooea

Nucleotidase Assay . . . . . . .
3'-AMPase Assay . . . . . . . .
Hyperchromic Assay . . . . . . .

ViSCOSity Assay o a e e a e o e

Other Enzyme Assays . . . . . . . . .

RNA Polymerase Assay . . . . . .

iv

Page

12
12
12
12

13
15
15
16
16

16

16
16
17
18
18
18
18

Page

Polynucleotide Kinase Assay . . 19
Alkaline Phosphatase Assay . . . 2O
Venom PhOSphodiesterase Assay. . 20
Protein Assays . . . . . . . . . . . . 20
Gel Electrophoresis . . . . . . . . . 20

Polyacrylamide Disc Gel Electro-

phoresis . . . . . . . . . . . . 20
SDS Gel Electrophoresis . . . . 21
Agarose Gel Electrophoresis . . 21
Isoelectric Focusing . . . . . . . . . 21
Ultracentrifugation Techniques . . . . 23

Sedimentation of DNA in Alkaline
Sucrose Gradients . . . . . . . 23

Sedimentation of DNA in Neutral
Sucrose Gradients . . . . . . . 23

CeCl Buoyant Density Banding . . 24

Model E Analytical Ultracentri-
fugation . . . . . . . . . . . . 24

Chromatographic Separation of 5'-Deoxy-

ribonucleotides . . . . . . . . . . . 24

Growth of Bacteria and Bacteriophages 27

M$WES ...,... ... ... ... ... ... 29
Wheat Seedling Nuclease Purification Proce-

dure . . . . . . . . . . . . . . . . . . . . 29

Type I Purification . . . . . . . . . 30

Type III Purification . . . . . . . . 31

Type II Purification . . . . . . . . . 34

Summary of Purification Procedure . . 35

Page

Protein Properties . . . . . . . . . . . . . 38

Sedimentation in the Model B Analytical
Ultracentrifuge . . . . . . . . . . . 38

Sedimentation through a Sucrose

Gradient . . . . . . . . . . . . . . . 40
SDS Gel Electrophoresis . . . . . . . 40
Sephadex G-100 Filtration . . . . . . 41
Isoelectric Point Determination . . . 44
Spectral Properties . . . . . . . . . 44

Multiple Ionic Forms of Wheat Seedling
Nuclease . . . . . . . . . . . . . . . 52

Mechanism of Action During Enzymatic

Catalysis . . . . . . . . . . . . . . . . . 58
Exo-and Endonucleolytic Cleavage . . . 63
3'-Nucleotidase Activity . . . . . . . (7
Kinetic Analysis of the Hydrolysis of
3"AMP and PAPA . . . . . . . . . . . 72
Base Preference at Cleavage Site . . . 79

Mechanism of Action on Native DNA . . . . . 82

Hydrolysis of Double-Stranded Synthetic

PolymeIS............... 81}
Limit Digests of Native DNA . . . . . 91
Mechanism of gh—l DNA Hydrolysis . . . 98

Acid Soluble Assays . . . . . . 102

Transcription of gh-l DNA Limit
Polymers . . . . . . . . . . . . 104

Buoyant Density Banding gh-l DNA
Limit Polymers . . . . . . . . . 107

Nucleotide Composition at 3' End
of gh-l DNA Limit Polymers . . . 107

vi

Effect of Temperature on

32P 5' End Labeling of gh-l DNA
Limit Polymers .

Hydrolysis . . . . .

DISCUSSION .

SUMMARY . .

APPENDIX A

APPENDIX B

APPENDIX C

BIBLIOGRAPHY

C O O O O O 0

Abbreviations
Growth Media

Calculations

vii

ADNA

Page

119
124
151
156
157
158
161

Table

II.

III.

IV.

VI.

VII.

VIII.

IX.

LIST OF TABLES

Comparison of various procedures
for the isolation of wheat seed-
ling nuclease. . . . . . . . . . . .

Molecular weight values and evi-
dence of purity for Type III enzyme.

Dephosphorylation rates on various
nucleotide substrates.,. . . . . . .

Base preference at cleavage site . .

Summary of molecular weights of
limit digest polymers from various
DNA source S" O O O O O O O O O O O 0

Acid soluble material produced
during digestion of 32P uniformly
labeled gh-l DNA by wheat seedling
nuclease at 22° and 37° C. . . . . .

Nucleotide composition at the 3'
end of uniformly 32F labeled gh-l
DNA limit polymers . . . . . . . . .

32F incorporated into gh-l DNA and
gh-l DNA limit polymers by poly-
nucleotide kinase. . . . . . . . . .

5' terminal nucleotide composition
of gh-l DNA limit polymers . . . . .

viii

Page

38

47

68
81

101

103

114

117

122

LIST OF FIGURES

Figure Page
1. Chromatography of 5'~deoxyribonuc1eo-
side monophosphates on a Dowex-l column 25
2. Chromatography of wheat seedling nu-

clease on a carboxymethyl cellulose
column following the phosphocell-
ulose purification step. . . . . . . . . 32

3. a. Chromatography of wheat seedling
nuclease on a carboxymethyl cell-
ulose column following the heat
stable purification step . . . . . . 36

b. Gel filtration of wheat seedling
nuclease on a Bio Gel P-60 column
following the carboxymethyl cell-

ulose step in Figure 3a. . . . . . . 36
4. Relative mobility of standard proteins
after SDS gel electrophoresis. . . . . . 42
5. Gel filtration of purified Type III
wheat seedling nuclease. . . . . . . . . 45
6. Isoelectric focusing of DNase and 3'-
AMPase activities. . . . . . . . . . . . 48
7. a. SDS gel electrophoresis of the
last three steps in the Type II
purification procedure . . . . . . . 53

b. SDS gel electrophoresis of the
last two steps in the TYpe III
purification procedure . . . . . . . 53

c. Polyacrylamide gel electrophoresis
of the purified wheat seedling
nuclease Type III on gels contain-
ing different percentages of acryl-
amide O O O O O O O O O O O O O O O O 53

l!

Figure Page

8. Plot of relative mobilities of
protein bands during electro-
phoresis on gels containing dif-
ferent percent of acrylamide . . . . . . 56

9. Isoelectric focusing of Type III
enzyme 0 O O O O C O O O O O O O O O O O 59

10. Ultraviolet absorbance scans of a
polyacrylamide gel following electro-
phoresis of Type III enzyme. . . . . . . 61

11. Time course for the hydrolysis of
polyadenylic acid by wheat seedling
nuclease Type II and muskmelon

nuclease O O O O O O O O O O O O O O C O 65
12. pH profiles for the hydrolysis of the

3'oribonuc1eoside monophOSphates by

wheat seedling nuclease. . . . . . . . . 70
13. a. Lineweaver-Burk plots for the

hydrolysis of 3'-AMP by wheat
seedling nuclease Type II show-
ing the inhibition by 5'-AMP . . . . 73

b. Lineweaver-Burk plots for the
hydrolysis of 3'-AMP by wheat
seedling nuclease Type II show-
ing the inhibition by 2',3'-CAMP
andApAeaeeaeeoeaeoooo 73

14. a. Lineweaver-Burk plots for the
hydrolysis of 3'-AMP by wheat seed-
ling nuclease Type II showing the
inhibition by NaCl . . . . . . . . . 76

b. Lineweaver-Burk plots for the
hydrolysis of pApA by wheat seed-
ling nuclease Type II showing the

inhibition by NaCl . . . . . . . . . 76
15. Time course for the hydrolysis of various
double-stranded DNA substrates by wheat
seedling nuclease Type II. . . . . . . . 84
16. Ionic strength and pH effects on the

initial velocities of hydrolysis of
poly d(A-T) and dDNA . . . . . . . . . . 87

Figure

17.

18.

19.

20.

21.

22.

24.

Page

Time course for the loss of acid

insoluble substrate during the

hydrolysis of native gh-l DNA,

denatured gh-l DNA and poly rA,U . . . . 89

Time course for the decrease in

molecular weight during the hydro-

lysis of native E, coli DNA by wheat

seedling nuclease Type II. . . . . . . . 93

Alkaline sucrose gradient sediment-
ation of A DNA and its reaction _
prOdUCts O O O O O O O O O O O O O O O O 96

Neutral sucrose gradient sedimentation
of gh-l DNA and its reaction products. . 99

RNA polymerase activity on a limit

digest of gh-l DNA after sediment-

ation through a neutral sucrose

gradient . . . . . . . . . . . . . . . . 105

a. Substrate saturation and Line-
weaver-Burk plots for the trans-
cription of gh-l DNA by gh-l phage-
induced DNA dependent RNA polymer-
ase. O O O C O O O O O O O O O O O O 108

b. Substrate saturation and Line—
weaver-Burk plots for the trans-
cription of gh-l DNA limit polymers
by gh-l phage induced DNA dependent
RNA polymerase . . . . . . . . . . . 108

a. Buoyant density banding of gh-l
DNA in a CeCl gradient . . . . . . . 110

b. Buoyant density banding of gh-l
DNA limit polymers in a CeCl
gradient 0 O O O O O O O O O O O O O 110

Dowex-l separation of nucleotides pro-

duced by degrading gh-l DNA limit polymers.
which had been labeled with 32P at the

5' termini, to the mononucleotide
leveleeeeeeeaaaeoaooaae 120

xi

INTRODUCTION

The ubiquitous presence of nucleolytic activities
in the cells of organisms in all life's phylogenetic levels
has been recognized for some time. Nucleic acids. the
target molecules for these activities. serve the important
function of maintaining and perpetuating the biological
information necessary for life. It is not surprising. then.
that nucleases have been the subject of a great deal of
interest to the biochemist. During the past two decades.
which saw numerous attempts by enzymologists to purify and
characterize manyenzymes of diverse nature. a large number
and variety of nuclease activities has been reported. These
nucleolytic activities have been characterized and cataloged
according to their hydrolytic and degradative properties (1).
until very recently. however. little attempt has been made
to ascribe these activities to particular cellular functions.
The molecular mechanisms and enzymatic involvement in such
subcellular phenomena as genetic recombination. DNA repli-
cation and repair. and restriction of foreign DNA molecules
has led to a renaissance of interest in nucleolytic enzymes.

Of these cellular processes. perhaps the one with the
most information concerning nuclease involvement is that
of DNA replication and repair. The early evidence indicating

l

2

the involvement of nucleolytic enzymes was based on the
results from three experimental approaches (2). The
first was the correlation between high levels of DNase
activities with the period of growth corresponding to
the fastest rates of DNA synthesis. Secondly. it was
pointed out that the infection of a host bacterial cell with
a phage usually resulted in the appearance of a variety of
nuclease activities even in the case where the host DNA
was not degraded. And. finally. the appearance of nucleo-
lytic activity associated with a DNA polymerase purified
to a single protein species indicates an intimate relation-
ship between the nucleolytic and polymerase activities.

Goulian (3) has recently reviewed the properties of
the nuclease activities associated with g. 92;; DNA poly-
merase. There appear to be two separate DNase activities:
one acting in the 5' to 3' direction and the other in the
3' to 5' direction. The 5' to 3' activity requires a double
helical structure for activity and a 5' OH or 5' phosphate
terminus provides an equally acceptable substrate. In con-
trast. the 3' to 5' activity is greater with single-stranded
DNA and requires the presence of a 3' OH group. The 3' to
5' degradation produces some di- and oligonucleotides.
Recently Kornberg 2;,31. (4) have succeeded in physically
separating 5' to 3' nuclease activity from E. 22;; DNA
polymerase molecule following proteolytic digestion. {A
hypothetical scheme has been proposed for the concerted
hydrolysis and polymerization of DNA by g, 22;; DNA

3
polymerase and is reviewed by Goulian (3). This scheme
postulates a spatial orientation for the catalytic sites
on the enzyme which is compatable with the overall polymeri-
zation reaction. The ability of the 5' to 3' nuclease to
remove segments of DNA in which the bases are not paired
suggests a repair role for g, 22;; DNA polymerase and.
indeed this may be the exclusive role for the enzyme.

The subject of DNA repair and its relationship to
nucleases has been reviewed by Howard-Flanders (5). The
excision of mismatched base pairs as well as pyrimidine
dimers from UV irradiated DNA suggests the presence of
endonucleases with the ability to recognize these parti-
cular sites on the DNA molecule.

In its broadest sense the biological process of
recombination has been defined as "any of a set of path-
ways in which elements of nucleic acid interact with a
resultant change of linkage of genes or parts of genes” (6).
Recombination has been most thoroughly studied and best
understood as a genetic phenomenon rather than a biochem-
ical process involving particular enzymes and pathways.
Nevertheless. certain authors have proposed various models
or schemes for recombination which postulate the involvement
of exonucleases. endonucleases. ligases and polymerases (6.7).
In its original genetic perception. recombination occurred
through a process of synapsis occurring at the same loci
on homologous regions of DNA followed by crossing over

to form the recombinant molecules. The phenomenon of

4

”site-Specific" recombination, which involves the integra-
tion and excision of temperate phage with the host genome,
has recently received considerable attention (8,9). The
proposed model for this process is based on evidence from
genetic studies with bacterial mutants and postulates

the recombination between specific but nonidentical regions
(phage and host attachment regions).

The final area of nuclease involvement to be reviewed
is that of the bacterial modification-restriction systems.
These systems are responsible for degrading foreign unmod-
ified DNA molecules which might enter a bacterial cell.

The modification and restriction activities appear to be
genetically linked and separate systems have been identi-

fied in five strains of E, 321;. as well as in several other
bacteria including Hemophilus influenza. The latter example
contains a restriction-like endonuclease but as yet no detect-
able modifying activity. A common feature of all the restric—
tion endonucleases is their ability to produce a few, speci-
fic double-stranded scisions in unmodified DNA molecules,
yielding limit fragments of different lengths.

A considerable amount of information about the restric-
tion endonucleases has recently been obtained (10). All
of the restriction endonucleases investigated to date require
Mg*+ and in the case of at least three of the enzymes, ATP
and S-adenosylmethionine are either required or provide
activating effects. The enzymes are generally large pro-

teins (MW 80-300,000) and subunits have been identified

5

in some cases. All of the restriction endonucleases invest-
igated thus far exhibit a pH optimum between 7-8. Further-
more they apparently lack the ability to catalyze hydrol-
ysis of single-stranded DNA.

Since restriction endonucleases produce such a limited
number of double strand scisions (0.01-0.l% of the phos-
phodiester bonds available) the question naturally arises
as to the nature of the substrate site recognized by the

enzyme. The cleavage site for the restriction-like endo-

 

nuclease from H, influenza has been studied in detail (ll,
12). The site is uniquely defined by a hexanucleotide
sequence of the following sort:

5'---PGPTPP;PPUPAPCP"-3'

3 ' ---pCpApP ugrPyprGp-"S '
where the arrows indicate the bonds cleaved. The striking
feature of this sequence is the two fold rotational axis
of symmetry perpendicular to the DNA helix. It was post-
ulated (11) that symmetry elements in the protein (possibly
at the subunit level) are able to recognize the base symp
metry in the DNA sequence.

Similarly, Hedgpath EEHEl- (13) have established
the base sequence recognized by the g, 321;. RI restriction
endonuclease:

5'---A/TPéPAPAPTPTPCPT/A---3'

3'---T/APCPTPTPAPARFPA/T---5'
Again a two fold rotational symmetry is noted. While the

H, influenza enzyme attacks diester bonds directly across

6
from each other, the RI endonuclease produces a staggered
cleavage resulting in cohesive ends. In addition it is
noted that although the RI enzyme is specific for the bond
adjacent to the 3' side of guanine, twelve of the sixteen
bases in the recognition site are either A or T.

While the aforementioned cellular processes are cer-
tainly not the only events involving nucleolytic activity,
they clearly are vital to the life of the cell. Other possible
functions for nuclease activities in the cells may involve
processing of both nuclear and cytoplasmic RNA molecules
as well as providing a general pool of nucleic acid come
ponents. The omnipresence and diverse functions of nucleases
coupled with the obvious need for a cell to maintain its
genetic integrity would seem to indicate the importance
of control and regulation mechanisms for the expression
of various nucleolytic activities. Apart from the known
examples of induction of nuclease activities following
viral infection little is known concerning the control of
nuclease activities already present in a normal cell.

It has been suggested (14) that control of intracellular
RNase activity could arise from a) spacial segregation,

b) complexing of the RNA to form a resistant structure,

c) complexing of the enzyme with an inhibitor, and d) Speci-
ficity for one form of macromolecular structure of RNA.

Most of the evidence centers on b and c. RNA in ribosomes
has been shown to be resistant to RNase attack. Finally.a

number of nuclease inhibitors have been found,some of which

 

1A list of abbreviations may be found in the appendix.

are proteins.

Wheat seedling nuclease, originally isolated in this
laboratory by D. Hanson (15), can be classified among other
nucleolytic enzymes by a number of catalytic properties.
The enzyme hydrolyzes the phosphodiester bond in RNA and
DNA producing 5' phosphoryl terminated oligonucleotides
and mononucleotides. In addition, the enzyme possesses
the ability to cleave the phosPhate moiety from 3'-nucleo-
side monophosphates. Wheat seedling nuclease appears to
cleave dDNA in an endonucleolytic manner while degrading
rRNA exonucleolytically (l6). Denatured or single stranded
DNA is hydrolyzed at a much faster rate than native helical
DNA .

Several other nucleases also demonstrate a definite
preference for single-stranded substrates and have been
isolated from lamb brain (17), sheep kidney (18), yeast
cells (19), Neurosppra crassa conidia (20), Staphylococcus

aureus (21), Aspergillus ogyzae (22), and mung beans (23).

 

These enzymes show several common properties in regards to
substrate specificity: a) they are all capable of hydro-
lyzing internal Phosphodiester bonds (endonucleases), b) the
the reaction products are 5' phosphoryl terminated oligo-
and mononucleotides except in the case of g, aureus which
produces 3'-P terminated products, and c) there is no speci-
ficity for the sugar moiety (is. DNA and RNA are both sub-
strates).

The single-stranded specificity of these enzymes

8
has found utility in several cases for use in hybridization
experiments where selective removal of nonhybridized areas
is desirable (24,20). In addition, the ability of these
enzymes to recognize differences in the ordered structure
of the substrate makes them potential tools for probing
nucleic acid structure. For example, sheep kidney enzyme
has been used to study the looped regions in tRNA (18).
Furthermore, it has been demonstrated that the enzyme from
.§. aureus can recognize A-T rich areas in native DNA (21).
This preference was ascribed to the fact that the dynamic
equilibrium (single-strandzsdouble-strand) for areas rich
in A and T is shifted to the left.

One of the single strand specific nucleases most
thoroughly studied is that isolated from mung beans (23,
25-29). This enzyme appears quite similar to the wheat
seedling nuclease and will be reviewed here in some detail.

Like the wheat seedling nuclease, the mung bean enzyme
is a) a 5' phosphate former, b) sugar unspecific, c) an
endodeoxyribonuclease and d) is capable of catalyzing the
hydrolysis of the phosphomonoester bond of 3'-AMP. In add-
ition, Johnson and Laskowski (27) have demonstrated a high
degree of enzymatic preference for single-stranded substrates
as opposed to the native or double helical structures.
Their most striking comparison is that for native and heat
denatured T. DNA. The initial velocities of hydrolysis of
these two substrates differ by 30,000 fold when measured

by a hyperchromic assay. The authors tentatively suggest

9
that the small amount of hydrolysis of native T4 DNA may be

due to single-stranded contamination. This is consistant
with the kinetics observed as well as the lack of decrease
in 820’w for the native substrate. Somewhat smaller differ-
ences are noted for the initial rates of native and heat
denatured forms of E, $211. and calf thymus DNA. using

the differences in buoyant density between native and denat-
ured DNA, it was demonstrated that T. DNA corresponding

to the denatured DNA peak on a OaCl gradient was select-
ively and completely removed from a mixture of native and
denatured forms by the mung bean enzyme without changing

the native DNA peak. The relative degree of susceptibility
of the substrate's secondary structure was tested by com-
paring rates of hydrolysis of a number of denatured substrates
which had been renatured to varying extents. These results
were also consistant with a very high level of specificity
of the enzyme for the single-stranded form of nucleic acid
polymer. The only exception noted by the authors to the
resistance of double stranded substrates was the synthetic
copolymeric duplex, poly d(AT) which was hydrolyzed at

rates comparable to denatured DNA. In a rather dramatic
experiment using high levels of enzyme at 170 C, it was shown
that mung bean nuclease could selectively cleave the linear
duplex,x DNA, near its center producing two undamaged halves
which were separable due to their difference in G plus C
content, hence buoyant density. At temperatures above

17° C this center-recognizing specificity was lost. The

10

authors point out that )xDNA has a well characterized A-T
rich region near its center (spanning 0.1 of the total
genome). This fact taken together with the susceptibility
of poly d(A—T) to mung bean nuclease led the authors to
coin the phrase ”region specific nuclease”.

The mung bean nuclease exhibits a chemical preference
for the rate of hydrolysis of the diester bonds adjacent
to the five naturally occurring bases in single stranded
nucleic acid polymers (28). The order of preference in
denatured DNA and riboheteropolymers was found to be
A>T(U) > C >G. This is the same order as that found for the
monophosphatase activity of mung bean nuclease on 3'-nucleo-
tide substrates. Loring g§,al. (26) working at a higher
pH found a different relative order - namely. A’G7 U >C.

The work to be described in this thesis represents
an attempt by the investigator to provide a general pic-
ture of the nature of the molecular events occurring during
the enzymatic catalysis by wheat seedling nuclease.
Theoretically. a true definitive description of the mole-
cular mechanism of any chemical reaction as complex as
the hydrolysis of a polynucleotide would require an exceed-
ingly large number of assignments of the coordinates of
all involved atoms in space and time. Therefore. any"mechan-
istic study“ represents an investigation yielding a qualita-
tive description. the detail of which is limited by the
amount of experimental data. The portion of this thesis

directed toward elucidation of the biochemical ”mechanism

ll
of action" of the wheat seedling nuclease, then is given
in this general descriptive sense.

It has been widely observed in the biochemical lit-
erature that protein structure plays an important part in
enzymatic function. With this structure-function relation-
ship in mind, wheat seedling nuclease was isolated in a highly
purified form and some of its physical and chemical pro-
perties are reported. Furthermore, some of this data has
led to the construction of a possible model for the enzy—
matic control of wheat seedling nuclease in the cell.

A third section of this thesis reports in a somewhat
detailed manner on the mode of enzymatic hydrolysis of dou-
ble stranded, native DNA by wheat seedling nuclease. The
special attention to this aspect of the reaction mechanism
seems warrented for several reasons. The overall biolog-
ical implications of the manner in which native DNA is
hydrolyzed by the wheat enzyme may relate to some of the
cellular functions for nucleases reviewed earlier in this
introduction. Furthermore, these studies indicate a poten-
tially important role for wheat seedling nucleases as a

probe of DNA structure.

EXPERIMENTAL PROCEDURE
Materials
Resins

Carboxymethyl cellulose cation exchange resin (0.07

meg/gm capacity, coarse mesh) was purchased from Sigma.

It was prepared before use by washing with 2 cycles of

0.5 M NaCl - 0.5 N NaOH and water, then with 0.1 N HCl and
water, with another cycle of 0.5 M.NaC1 - 0.5 N NaOH and
finally by extensive washing with water.

Cellulose phosphate cation exchange resin (0.8 meq/gm
capacity, coarse mesh) was purchased from Sigma and treated
prior to use with alternate washings of 0.1 N KOH and water
until it was colorless.

Dowex 1-X4 was purchased from Bio Rad Laboratories
as analytical grade Ag l-X4, 100-200 mesh, chloride form.
Before use it was subjected to 3 cycles of l N NaOH and
l N HCl and then washed extensively with water. DEAE Sephadex
A-25 and Sephadex G-100 were products of Pharmacia and were
used without additional treatment. Bio-Gel P-60 (50-150
mesh) was obtained from Bio Rad Laboratories and DEAE cell-
ulose (DE-52) was a product of‘Whatman.

Proteins

The proteins used as standards in sucrose gradient

sedimentation and SDS gel electrophoresis were egg

12

13

ovalbumin, Grade III from Sigma; cytochrome C, Type III
from Sigma; trypsin, Type III from Sigma; and hemoglobin
which was a gift from Dr. A. Morris.
Enzymes

The following enzymes were obtained commercially
and used without further purification: adenosine deaminase,
Type I from Sigma; DNase I and DNase II; DPFF and HDAC
respectively from Worthington; and micrococcal nuclease,
Grade IV from Sigma.

Alkaline phOSphatase was purchased from Sigma as
Type III and was carried through an additional purification
by chromatography on a DEAE cellulose column (30). Venom
phosphodiesterase was purchased from Worthington (VPH)
and trace amounts of 5'-nuc1eotidease were removed by the
method of Laskowski (31). 5'-AMP amino hydrolase was either
a gift of Dr. C. Suelter or was purified from rabbit muscle
by the method of Suelter et a1, (32). Muskmelon nuclease
was prepared in this laboratory by Dr. L. Muschek. The
RNA dependent DNA polymerase induced by infecting Pseudomonas
putida with the gh-l phage was a gift of H. Towle of this
department. Polynucleotide kinase was purified from T4
infected.§,‘ggli B through the phosphocellulose step after
the method of Richardson (33).
Nucleic Acids

The DNA routinely used as a substrate for DNase activity
was highly polymerized salmon sperm DNA (Sigma Type III -

sodium salt). Ribosomal RNA was prepared in this lab by

14
the method of Cresfield (34). High molecular weight
g, ggli_DNA was made by Dr. D. Hanson2 by a modification
of the method of Berns and Thomas (35). The polyribohomo—
polymers, poly d(A-T), and poly dG - poly dC were purchased
from Miles Laboratories. Poly rA,U was a gift from Dr. J.
Boezi. The 5' ribo- and deoxyribonucleoside monophosphates
were purchased from Sigma as was 3', 5' - cyclic AMP.
The 3'- and 2'-ribonuc1eoside monophOSphates were products
of PL Labs. The 3'—deoxyribonuc1eoside monophosphates
were a gift from Dr. D. Hanson.

DNA from the bacteriophages T4, T7, A and gh-l was
prepared by the following procedure. SDS was added to a
phage suspension (concentration of phage was adjusted to
give 10-25 Optical density units at 260 mm per ml) to give
a final concentration of 0.25%. The suspension was then
heated to 600 C for 3 minutes. The DNA was extracted 3
times with freshly distilled, water-saturated phenol.

The aqueous phase was then extensively dialyzed against
50 mM NaCl, 10 mM tris-HCl (pH 8) until all of the phenol
had been removed. Q -X 174 RF DNA was the generous gift
of Dr. R. Wells, Department of Biochemistry, university
of‘Wisconsin.

The di- and trinucleotides, pApA and pApApA were
made by digesting poly A with muskmelon nuclease. The
reaction mixture contained 20 ml 0.05 M succinate buffer,

pH 6.0, 5 ml poly A (212 optical density units at 260 nm)

 

2 . . . .
Research Division, veterans Administration Hospital,
Bedford, Massachusetts.

15

and 0.11 ml muskmelon nuclease. After incubating at 37° C
for two hours, the reaction was stopped by heating at
950 C for 3 minutes. The mixture was then applied to a
DEAE Sephadex A-25 column (2.5 x 60 cm) which had been
equilibrated with 0.14 M triethylammonium bicarbonate
(TEAB), pH 8.0. The column was washed with 1 liter of 0.14
TEAB and then an 8 liter gradient of 0.14-0.46 M TEAB was
applied to the column. A flow rate of 1.0 ml/min was
maintained and 20 minute fractions were collected. Three
peaks of absorbance at 260 nm were observed. They were
identified as pA, pApA and pApApA by comparing the
inorganic phOSphate to nucleotide ratio after treatment
with alkaline phosphatase. The pApA and pApApA peaks
were pooled and flash evaporated to dryness three times.
The residue from the last evaporation was dissolved in
water and passed through a phosphocellulose column at
pH 7.0 to remove traces of triethylamine.

ATP labeled in the X position with 82P was made
by a modification (33) of the method of Glynn and
chappell (36).
Bacteria and Bacterigphages

E,‘ggli B, Pseudomonas putida and gh-l bacteriophage
were supplied by Dr. J. Boezi. The temperature sensitive
mutant of E, 991; (CgooACIt8857) containing A prophage and
T4 bacteriophage were gifts from Dr. L. Snyder, Department
of Microbiology, Michigan State University. T7 bacterio-

phage was supplied by Dr. D. Hanson.

16

Chemicals

Cesium chloride (optical grade) was purchased from
Schwarz/Mann. Ethidium bromide was a product of Calbiochem.
Guanidine—HCl (ultra pure) was a product of Schwarz/Mann.
Agarose used in gel electrophoresis was obtained from Sigma
as "electrophoresis grade." Acrylamide was a product of
the Canalco Company.
mass.

The wheat used as a source of wheat seedling nuclease
was Genesee soft white wheat (either certified or one year
removed from certification) which had been treated with

a mercurial fungicide.

Methods

Assays for Activity of Wheat SeedlinggNuclease

Standard Agid:Soluble_QNase and RNase Assay unless
otherwise stated, all assays for RNase and DNase activities
of the wheat enzyme were performed with the acid-soluble
technique of D. Hanson (15). The precipitating reagent
is La(N03)3 - HCl (0.02 M La(N03)3, 0.2 m HCl) which is
mixed with an equal volume of reaction mixture. A unit
of activity is defined as that amount which catalyzes in
10 minutes the formation of lanthanum acid-soluble material
with an A230 of 1.0 per ml of incubation mixture under
standard conditions.

Nugleotidase Assay In order to study the nucleotidase

activity of wheat seedling nuclease, two different assay

17
techniques were used. A fixed time phosphate assay pro-
cedure was used to evaluate various substrate analogs as
well as determine pH optima for certain substrates. The
procedure measures the formation of a colored molybdate
complex and is a modification (15) of the method of Dreisbach
(37). One unit of activity is defined as the amount of
enzyme which liberates l Jxmole of Pi per m1 of incubation
mixture in 15 minutes under standard conditions.

3'vAMPase Assay The second method was developed as

 

a continuous Spectrophotometric method and used to study

the kinetics of the release of phosPhate from 3'-AMP.

The coupling enzyme used in the assay was adenosine deaminase.
This enzyme catalyzes the hydrolysis of the amino group at
the 6 position of adenosine converting it to inosine which
has a considerably smaller molar absorbtivity at 265 nm.
Adenosine deaminase is specific for adenosine and will not
deaminate 3'-AMP (38). The enzyme's Km, broad pH optimum
and high catalytic turnover number (38) make it an effi-
cient coupling enzyme in this reaction. The difference in
the extinction coefficients for adenosine and inosine at

265 nm is such that a difference of 1.0 A253 unit equals

the conversion of 0.14 famoles adenosine per ml to inosine.
The kinetic assays on 3' AMP were performed at 30° C, pH 6.0
in 0.01 M cacodylate buffer. Under these conditions 1 unit
of 3'-AMPase activity is defined as the hydrolysis of l
Jamole of 3'-AMP per minute. In all cases the amount of

wheat seedling nuclease used was such that the rate of

18
hydrolysis fell on the linear portion of the enzyme vs. rate
curve. Furthermore, the amount of adenosine deaminase used
was always such that additional deaminase produced no change
in the reaction rate.

Hyperchromic Assay. In certain instances the increase
in optical density at 260 nm due to the hyperchromic pheno-
menon was used to measure the rate of hydrolysis of nucleic
acid polymers by wheat seedling nuclease. These measure-
ments were made in a Beckman DB spectrophotometer with the
aid of a Sargent model SRL recorder. Rates were observed
in the 0.5 to 1.5 OD range and except when noted, were linear
for at least the first minute. Initial rates are reported
as the increase in A260 units per minute.

Viscosity Assay A loss of viscosity was used to
measure the reduction in molecular weight of E, 9211 DNA
during the time course of hydrolysis by wheat seedling
nuclease. Viscosities were determined with a Contraves
Low-Shear Rheometer type LSlOO (Contraves AG, Zurich)
with a minimum shear rate of 0.07 per second. At the times
specified 0.35 ml of reaction mixture were placed in the
viscometer. Viscometer values at various shear rates were
recorded and essentially no shear dependence was noted at
the lower shear rates. Molecular weights were calculated
from the empirical equations of Eigner (39) where k, the
concentration dependency constant was 0.6.

Other Enzyme Assays

RNA Polymerase Assanghage-induced, DNA-dependent

19

RNA polymerase from Pseudomonas putida was assayed accord-
ing to the procedure of H. Towle. The assay procedure con-
sists of measuring the amount of 3H- labeled CTP incorporated
into acid-insoluble form in 15 minutes at 30° C. The reac-
tion mixture contained 25 ,ul of prereaction mix (200 ,ul
1 M tris-HCl, pH 8, 50 ,al 1 M M9012, 50 ,ul 0.1 M dithio-
threitol, 200 111 10 mg/ml BSA, 100 [ll of nucleoside tri-
phosPhates -0.02 M in each nucleotide, 100,/A1 3H - CTP
0.5 Ci/ml, 300/41 H20), 10 /UL1 DNA substrate and 10 3:11
polymerase. The reaction was terminated by the addition
of 0.1 ml 0.1% SDS, 2 drOps of salmon Sperm carrier DNA
and 5.0 ml cold 10% TCA - 1% PPi. After standing for 5
minutes in ice, the material was filtered and rinsed 3 times
with 10% TCA - 1% PPi on a millipore filter. The filters
were dried, covered with scintillation fluid and counted
in a liquid scintillation counter. One unit of activity
equals 1 nmole osz-CTP made acid-insoluble in 10 minutes.

Polynucleotide Kinase Assay. Polynucleotide kinase
was assayed by the method of Richardson (33). The assay
measures the conversion of radioactivity from X'- 32P-ATP
into acid-insoluble form. The substrate used was made
by digesting salmon Sperm DNA with micrococcal nuclease.
A unit of polynucleotide kinase activity is defined as
the amount catalyzing the production of a m/Amole of acid-
insoluble 32F in 30 minutes at 37° C.

Alkaline Phosphatase Assay' The method used to assay

alkaline phoSphatase was that of Garen and Levinthal (30)

20
where the change in absorbency at 410 nm corresponds to
the release of p-nitrophenol from p-nitrophenyl phosphate.
One unit liberates one‘ximole of p-nitrophenol per minute
at 25° C.

Venom Phggphodiesterase Assay Venom phosphodiesterase
was assayed using the procedure of Laskowski (31). One
unit equals the amount of enzyme needed to liberate one
jimole of p-nitrophenol per minute from p-nitrophenyl
phosphate at 25° 0.

Protein Assays

Protein determinations were performed after the
method of Lowry pg 31. (40) or by the turbidometric method
using tannic acid as a precipitating reagent (41). The
latter method has several advantages - namely. tris buffer
and cysteine do not interfere. the assay is linear from
20-100 llgms/ml of protein. and finally. determinations
can be made in ten minutes. BSA was used as a standard

protein with both methods.

Gel Electrophoresis
Pglyacrylamide Disc Gel Electrophgresis Polyacryl-

amide disc gel electrophoresis was performed by the method

of Ornstein (42) and Davis (43). Except where otherwise
noted. 5 cm of 7 Percent running gel with 2 cm of stacking
gel on top was polymerized in a 0.5 cm (inside diameter)
glass tube. Protein (50-100.;Lgms) was layered on top of

the gels in 20% sucrose in a volume less than 0.1 ml. Bromo-
phenol blue was used as a tracking dye and electrophoresis

was stopped when the dye band was just off the gel,

21
(about 90 minutes). Gels were stained for protein with
Coomassie Blue, destained and stored in 10% acetic acid.

SDS Gel Electrophoresis SDS gel electrophoresis was
performed following the procedure of Weber and Osborn (1H0.
Protein solutions were prepared in 1% SDS and 1% 2-mercapto-
ethanol and heated at 1000 C for 10 minutes prior to electro-
phoresis. Ten-cm gels containing 5% acrylamide (w/v) were
used. Electrophoresis was carried out at room temperature
at 8 ma/tube for 3 hours. Under these conditions the dye
band had migrated through approximately one half of the
gel. The smallest polypeptide moved about 10% farther than
the dye band. Protein was stained and destained as des-
cribed previously.

Agarose Gel Electrgphoresis Electrophoresis of DNA
on agarose gels in the presence of 0.5 3Ug/m1 of ethidium
bromide was accomplished following the procedure of J.
Sambrook g£_31, (45). The DNA appeared as fluorescent bands
under short-wave UV illumination. The gels were photo-
graphed with Polaroid type 55 P/N film after removing the
illuminating short-wave UV with a Kodak No. 23A red filter.
Isoelectric Focusing

Two different procedures were employed for isoelec-
tric focusing experiments. The first procedure used to
determine the isoelectric point of the wheat seedling
nuclease activities was carried out in an LKB Model 8101
electrofocusing column (total capacity 106 ml) which con-

tained a pH gradient of ampholyte from 3.0-10.0. A

22
potential of 300 volts was applied to the column for 48
hours using a Krohn-Hite Model U4sz40 constant voltage
power supply.

The following procedure (46) was used for the
isoelectric focusing experiments conducted on the puri-
fied Type III wheat seedling nuclease. A column (154 mm x
10 mm) containing ampholites giving a final pH gradient of
3-6 was used. A 1 cm plug was formed in the bottom of
the column with a mixture containing 1 percent agar and
3 percent ethanolamine. To the upper part of the column
3 percent phosphoric acid was carefully layered and the
complete upper reservoir was then filled with 3 percent
phosphoric acid. The lower reservoir was filled with
3 percent ethanolamine. In a cold room a 4° C and with
the use of a Heathkit constant voltage power supply. 200
volts were applied to the column with the upper reservoir
being positive and the lower reservoir being negative.
After the current had dropped to less than 10 percent of the
original value (about 4 hours). the column was fractionated
by removing 0.2 m1 fractions off the top with a spring-
1oaded syringe fitted with a piece of polyethylene tubing.

Ultracentrifugation Technigues
Sedimentation of DNA in Alkaline Sucrose Gradients

Alkaline sucrose gradients were prepared by forming a 4.5
m1 linear gradient of 5-20% sucrose which was 0.9 M

NaCl and 0.1 M NaOH. DNA samples (0.1 ml) were allowed

to set 30 minutes in 0.9 M NaCl. 0.1 M NeOH and then

23

layered on top of the gradient with polyethylene tubing
(1.0 mm I.D.) and a syringe microburet. The tubes were
placed in a SW-39 rotor and spun in a Beckman Model L-2
centrifuge for 3 hours at 35.000 RPM at 20° 0. In the
case of 32F labeled DNA. 1-5 ,ugms DNA was applied to the
gradient and 30 fractions collected. The fractions were
either counted directly as Cerenkov radiation or were TCA
precipitated. filtered and counted in scintillation fluid.
When unlabeled DNA was sedimented. 15-30 ,ugms DNA was
layered on top of the gradient and 25 fractions collected.
Optical density at 260 nm of the individual fractions was
measured in 0.2 m1 cuvettes with a Gilford spectrophoto-
meter. The following formula was used to correlate S
values with molecular weight - S=0.0528 x MW°u°°.

Sedimentation of DNA in Neutral Sucgose Gradients
Sedimentation of DNA in neutral sucrose gradients was
performed in an analogous manner except that the DNA was
not treated with NaOH and NaCl prior to centrifugation.
The 5-20% sucrose gradient contained 1.0 M NaCl and 10 mM
tris-H01. pH 8.0. The gradients were spun at 50.000 RPM
for 3 or 3.5 hours at 5° C. Uhder these conditions

s=0.0882 x hw‘jué.

CsCl. Buoyant Density Banding To form a gradient of
CsCl. 4.5 m1 of CeCl with a refractive index of 1.4000
were placed in a centrifuge tube. After layering the DNA
sample (0.1 ml) on top of the gradient. the tubes were spun
at 32.000 RPM for 63 hours at 10° C in a fixed-angle rotor.

24
number 40. Forty fractions were collected and the refrac-
tive index was determined on every fourth fraction.

Model E Analytical Ultracentrifugation The sedi-
mentation coefficient for wheat seedling nuclease was
determined from sedimentation velocity analysis in a
Spinco Model E analytical ultracentrifuge. Protein and
solvent. respectively. were placed in the two chambers of
a 30 mm double sectored cell. An AN-E rotor number 414-V
was spun at 50.714 RPM at a temperature of 9° C. Photo-
graphs of the boundary pattern were taken at either 8 or
16 minute intervals.

Chromatographic Sepayation_of 5'-Deoxy;;bonucleotide§

Separation of the 5'-dNMP's was accomplished by
chromatography using Bio Rad AGl x 4 (100-200 mesh. Cl-
form) resin. The resin was washed with 3 cycles of 1 N
acid. water and base and then washed extensively with
water prior to use. A column (1.1 x 12 cm) was then
packed and washed with 200 ml of 2 mM 801. After the
sample was applied to the column. a 1.0 ml/min flow rate
was maintained with a Polystaltic pump and 5 minute fractions
were collected. Figure 1 shows a typical separation. The
sample applied to this column was comprised of 0.02 ml 1 M
tris-RC1. pH 7.8. 0.05 ml of each of the 5'-NMP's (10 mg/ml).
0.10 ml 0.1 M Mg012 and 0.1 ml 0.7 M glycine buffer. pH 9.2.
This mixture was diluted to 20 ml with water before appli-
cation to the column. Elution of the 4 nucleotides was then
accomplished with 3 mM HCl until pdC was off the column.
then with 5 mM HCl until pdA had been eluted and finally with

25

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26

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27

10 mM HCl which eluted pdG and pdT from the column. The
peaks were identified by comparing the absorbancy at 250,
260 and 290 nm with known spectra for the nucleotides in
acid.
Growth of Bacteria and Bacteriophages

T4r+ phages were grown3 at 37° C by inoculating a
culture of E, 22;; B cells (108 cells/ml in the logarithmic
phase of growth) with T4r+ phages at a multiplicity of
infection of 0.01. After shaking the infected cells at high
speed for 2 hours, lysis was completed with an additional
incubation for 4 hours at low shaker speed. Lysis was
followed by monitoring the absorbance at 650 nm. Phages
were purified by 2 series of differential centrifugations
(DNase I was added before the last low speed spin) and then
by banding in a step gradient of 20-70% saturated CeCl.
The purified phages were dialyzed against decreasing NaCl
concentration starting with 2 M NaCl and ending with 0.5
M NaCl.

Lambda phages were made by growing a culture of
temperature sensitive mutants of E, ggli, CgooACIt8875
to 0.5 A550 units at 30° C. The growth flask was placed
in a 42° C water bath for 15 minutes. It was then shaken
vigorously at 42° C for 1-2 hours until the A550 was 0.2.
DNase was added and the phages were purified by differential
centrifugation at 17,000 x g for 10 minutes after which

the supernatant fluid was spun an additional 5 hours to

 

3See appendix for the composition of the various
growth media.

28

pellet the phages. The resuspended phages were subjected
to 10 minute and 5 hour Spins and the resuspended phages
were then further purified by banding in a step gradient
of 20-70% saturated CeCl. The phage suspension was dialyzed
against 0.5 M NaC1, 0.01 M tris-HCl pH 8.0 before the DNA
was extracted.

32F uniformly labeled gh-l DNA was made by growing
Pseudomonas putida in a low phOSphate media at 32° C to
0.2 A560. 32P was then added (3 mCi per 500 m1 growth
medium) and 30 minutes later gh-l phages were added at a
multiplicity of infection of 0.2. After lysis was complete,
a few drops of chloroform.were added and the lysate stored
overnight at 4° C. The lysate was first spun at 16,000 x
g for 10 minutes. Then the supernatant fluid was spun at
16,000 x g for 3 hours. The pellet was resuspended over-
night in 10 mM tris-HCl (pH 8) plus 0.2 M NaCl. After
another series of spins, the resuspended phages were passed
through a DEAE cellulose column at room temperature. The
column (2.5 x 10 cm) had been previously equilibrated with
10 mM tris-HCl (pH 8) plus 0.2 M NaCl. The fractions con-
taining the phage particles were collected and concentrated

by centrifugation before extracting the DNA.

RESULTS

Wheat Seedling Nuclease Purification Procedure

In order to conduct most of the experiments reported
in this thesis, wheat seedling nuclease was required in
relatively pure form with moderate yields of enzyme.

In addition, since the enzyme may possess utility as a

tool for research in other laboratories, expediency and
ease of preparation were also considerations. In order

to meet these requirements, several purification procedures
were developed during the course of this investigation.

As a starting point, the original isolation procedure

of D. Hanson (15) was used. This procedure consisted

of six steps. In the first step, the germinated

wheat seedlings were homogenized through cheese cloth.

In the next step the liquid portion of the crude homogenate
was subjected to precipitatiOn by a 57% saturation with
(NH4)ZSO.. A second treatment with 75% (NH4)ZSO4 pre-
cipitated most of the enzyme activity. In a likewise
manner, the third step consisted of a double precipita-
tion with ethanol. The fourth step, the heat stable step,
involved heating the material from the ethanol step to

75° C for 10 minutes and discarding the resulting

29

3“
precipitate. The next step was chromatography on a phos-
phocellulose column (PC step). The sixth and last step
in the original procedure was chromatography on a Bio-

Gel P-60 column.

Type I Purification

Initial efforts were centered on minor modifications
of the original isolation procedure outlined above and
resulted in the Type I purification procedure. Centri-
fugation of the crude homogenate at 10,000 x g for 15
minutes instead of squeezing through cheese cloth was
found to result in larger yields of enzyme. Typically
about 100,000 units of DNase activity and 100 grams of
protein were present in the crude homogenate which repre—
sents a three fold increase in activity and 50% increase
in protein.

Ammonium sulfate was added by an automatic contin-
uous shaking process during the (NH.)gSO. precipitations.
The pellet following the second (NH.)ZSO. precipitation
was dissolved in 0.05 M.NaAc, pH 4.5, 2 mM cysteine and
1.0 mM zinc acetate and stored overnight at 4° C with
no loss of DNase activity. This modification is a con-
venience since the preparation can be stopped after about
8 hours instead of the more than 12 hours previously
required to reach the heat stable step. The protein
collected and pooled from the final P-60 column step was
concentrated by pressure dialysis on an Amicon ultrafil-

trator (UMelO filter)which resulted in no loss of

31
activity. This material was dialyzed against 0.05 M.NaAc,
pH 5.0, 0.1 mM Zn(Ac)2 and 0.05% 2-mercaptoethanol and
was stable for months at 4° C. This purification pro-
cedure (Type I) involved the same six steps as the ori-
ginal procedure - namely, crude homogenate, (NH4)2SO.
step, ethanol step, 75° C heat treatment, phosphocellulose
chromatography and chromatography on a P-60 column and
required 5 days. This procedure was used through the heat
stable step as a starting point for the Type II purifica—
tion procedure and through the phosphocellulose step for

the Type III procedure.

Type III Purification

It was noted in batch studies with various ion
exchangers that the wheat seedling nuclease had a very
low binding affinity for carboxymethyl cellulose at pH
5.0. This property was utilized in the Type III puri-
fication procedure. The pooled fractions from the phos—
phocellulose step of Type I purification were concentra-
ted to 10-20 ml by ultrafiltration on a UMelO filter and
dialyzed overnight against two changes of 0.01 M.NaAc,
pH 5.0, 0.1 mM Zn(Ac)2 and 1.0 mM cysteine (CMC buffer).
This material (about 11,500 DNase units and 70 mg protein)
was applied to a carboxymethyl cellulose column and elu-
ted at room temperature with a 600 m1 linear gradient
of increasing NaAc concentration as shown in Figure 2.

As can be seen from the figure, the three activities

32

 

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33

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34
associated with wheat seedling nuclease eluted in the
early fractions while the bulk of the protein eluted in
two later peaks. The fractions containing the enzyme
activity were pooled and concentrated to approximately
1 ml by ultrafiltration on an Amicon UM-2 filter and
dialyzed against 0.05 M NaAc, pH 5.0, 0.1 mM Zn(Ac)2 and
0.05% 2-mercaptoethanol. Over 40% of the enzymatic act—
ivity and less than 0.8% of the protein applied to the
column was collected in the pooled fractions represent-
ing an increase in the specific activity of over 62 fold.
The activity of this preparation remained stable for sev-
eral months when stored at 4° C. This procedure yielded
wheat seedling nuclease in its purest form and was used
for the studies in the "protein properties" section ex—

cept where noted.

Type II Purification

A slightly less pure preparation (Type II) of wheat
seedling nuclease requiring 3 days to prepare was used
for most of the "mechanism of action" studies. In this
procedure, material from the heat stable step of the
Type I procedure was concentrated by ultrafiltration to
10-20 ml on an Amicon UMrlO filter and dialyzed overnight
against two changes of CMC buffer. This dialyzed solution
(about 16,000 DNase units and 120 mg protein) was applied
to a carboxymethyl cellulose column and eluted at room

temperature with a 600 ml linear gradient of increasing

35

NaAc concentration as shown in Figure 3a. The pooled
fractions of enzyme activity were concentrated and dia—
lyzed as described for the Type III procedure and were
also stable at 4° C for several months. Greater than

55% of the DNase activity and less than 2% of the protein
applied to the column was recovered in the pooled frac—
tions. As a result.this purification step represents

a 31 fold increase in the specific activity of the enzyme.
The Type II purified wheat seedling nuclease contained

no detectable 3'-nuc1eotidase or 5'-nucleotidase activity
at pH 5 or pH 7. In addition no hydrolysis of p-nitro-
phenyl phosphate or bis-p-nitrophenyl phosphate was noted
at pH 5 or pH 8. In an experiment to be described later
measuring acidosoluble material released from 32P labeled
native gh-l DNA, a small amount of a nonspecific exode-
oxyribonuclease was detected. In this case all of the
contaminating activity was removed by passing the enzyme
through a Bio Rad P-60 gel column as shown in Figure 3b.
The figure also shows a roughly uniform specific activity
across the elution peak indicative of a relatively high

degree of purity of the enzyme.

Summary of Purification Procedures

Table I compares the properties of the various
purification procedures. The numbers listed are typical
values based on at least three separate preparations.

Normally 3.5 kilograms of wheat were used as the starting

Figure 3a:

Figure 3b

36

Chromatography of wheat seedling nu-
clease on a carboxymethylcellulose
column following the heat stable puri-
fication step. Carboxymethylcellulose
was prepared as described in Materials.
A column (2.5 x 25 cm) was equilibrated
with 0.1 mM Zn(Ac)2, 1.0 mM cysteine
and .01 M NaAc, pH 5.0. The protein
was prepared and applied as described
in the text. A 600 m1 linear gradient
of increasing NaAc concentration (0.1
mM Zn(Ac)2, 1.0 mM cysteine, .01 M
NaAc, pH 5.0 to 0.1 mM ZnAc, 1.0 mM
cysteine, 0.3 M NaAc, pH 5.0) was used
to elute the enzyme. Fractions of 7
ml were collected and every second
fraction was assayed for protein (as
absorbance at 650 nm),o o and
DNase activity (as absorbance at 260
nm),a e .

 

 

Gel filtration of wheat seedling nu-
clease on a Bio Gel P-60 column follow-
ing the carboxymethylcellulose step

in Figure 3a. A column (2.3 x 46 cm)
was equilibrated with 0.1 mM Zn(Ac)2,
0.1% 2-mercaptoethanol, and .025 M
NaAc, pH 5.0. This same buffer was
used to elute the enzyme at a flow
rate of 0.8 ml/min. Fractions of

5 minutes were collected and each
fraction was assayed for protein
(absorbance at 650 nm) , 0———-o and
DNase activity (absorbance at 260 nm),
a a. The specific activity of the
eluted enzyme is plotted as DNase act-
ivity (absorbance at 260 mm) per mg/ml
of protein.

 

j
on

I
'0

 

F
I;

.r
N
Abs a! 2.0mm

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38

Table I

Comparison of various procedures for the isolation
of wheat seedling nuclease

 

 

 

Total Total Specific Fold
Procedure protein activity activity purifiedc YieldC
mg units units/mg %_—
Originalb 10.5 4,400 418 839 12.2
Type 1 13.0 15,000 1,150 1,150 15.0
Type II 2.2 9,000 4,090 4,090 9
Type III 0.5 5,000 10,000 10,000 5

 

aThese data are based on assays of the final purification
step of the procedure where the starting material was 7
kilograms of wheat seedlings. The standard DNase assay
was used to determine enzyme activity and, in the case of
procedures I-III, the data represent typical values of 3
or more purifications.

bThe original procedure is that of D. Hanson (15).

cFold purified and percent yield are calculated by comparing
the final purification steps with the crude homogenate.

39

material and two preparations were combined after the
heat stable step. The original isolation procedure
requires 5 days which included 12-18 hours at the begin-
ning before the enzyme is stable enough to store over-
night. The Type I procedure also requires 5 days but can
be stored overnight after about 8 hours. The Type III
procedure produced the purest enzyme and was judged to
be greater than 90-95% pure (see "Protein Properties"
section). This procedure requires 4 days for completion.
The Type II procedure was judged to give protein which
was greater than 80% pure (see the "Protein Properties"

section) and took 3 days to complete.

Protein Properties

In order to study some of the physical and chemical
properties of wheat seedling nuclease, it was necessary.
to establish the degree of homogeneity to which the en-
zyme had been purified. The Type III preparation was
used for these studies and several criterion were used

to estimate its purity.

Sedimentation in the Model E Analytical Ultracentrifuge
Wheat seedling nuclease Type III was sedimented

in the analytical ultracentrifuge as described in Methods.

A single symmetrical boundary was observed during the

sedimentation. The $20,w value which was calculated

40
from the sedimentation velocity data is 3.59. Assuming
that the enzyme is roughly Spherical in shape, this 820,w
value corresponds to a protein of approximately 43,000

daltons.

Sedimentation through a Sucrose Gradient

Type III enzyme (0.14 mg) was sedimented through
a 5-20% sucrose gradient in the swinging bucket rotor
of an L-2 Beckman ultracentrifuge following the proce-
dure of Martin and Ames (47). Centrifugation was done
at 4° C and stopped after 24 hours. Fractions were col-
lected and assayed for protein and DNase activity. A
single,coincident peak of protein and activity was found.
The Specific activity of the peak remained roughly con-
stant in all the fractions. Wheat seedling nuclease sedi-
mented to the same fraction number as egg ovalbumin (MW:-B
45,000) which was included in another centrifuge tube

in the same experiment.,

SDS Gel Electrophoresis

Acrylamide gel electrophoresis in the presence
of SDS was performed on protein samples from the last
three steps in the Type II purification procedure and
protein samples from the last two steps in the Type III
procedure as outlined in Methods. Typical gels are shown
in Figure 7a and 7b. The slowest moving band represents

the wheat seedling nuclease. As can be seen in the figure,

41
there are two other major protein bands after the heat
stable step. The CMC step removes most of these contam-
inating proteins and the band corresponding to the wheat
seedling nuclease is judged to comprise better than 80%
of the protein on the gel. The last step in the Type
II procedure, P-60 chromatography, increases the estima-
ted purity to better than 90%. The PC step of Type III
purification removes a significant amount of one of the
two contaminating bands present after the heat stable
step. The last step in the Type III procedure,CMC chro-
matography, results in a purification of wheat seedling
nuclease judged to be greater than 90-95% pure.

Figure 4 shows the relative mobility of several
standard proteins on SDS gels plotted against the log-
arithm of their molecular weights. When the relative
mobility of the single major band of the purified Type
III enzyme is compared to this standard curve, its mole-
cular weight is 43,500. Since this value is similar to
that derived from other methods (where conditions are
not expected to disrupt the subunit structure of proteins
like the conditions during SDS electrophoresis), wheat
seedling nuclease appears to be a protein comprised of

a single polypeptide chain.

Sephadex G-100 Gel Filtration
As a final criterion fOr estimating the homogen-

eity of the Type III purified enzyme, wheat seedling

Figure 4:

42

Relative mobility of standard proteins
after SDS gel electrophoresis. Relative
mobility refers to the distance moved
by the protein divided by the distance
moved by the dye band. Wheat seedling
nuclease relative mobility was 0.62
which corresPonds to a molecular weight

of 43,500.

4.50

4.25

leg MW

4.00

43

    
 
 
 
  
  
   

Egg Owclbumln (MW=43.000)

,Wlleul
Nuclease

Trypaln (MW= 23.000)

Haemoglobin

(MW:15,000)

Cylochrome C
(MW . 11.000)

. .- . 1.0 1.1
Role! Ive Moblllly

1.2

44

nuclease was chromatographed on a Sephadex G-100 column.
The result is shown in Figure 5. All of the protein
eluted in a single peak with the enzyme activity between
1 and 2 times the column void volume. The specific acti-
vity remained constant throughout the peak. The various
techniques used to estimate the Size and purity of wheat

seedling nuclease Type III are summarized in Table II.

Isoelectric Point Determination

The isoelectric point for the 3'-AMPase and DNase
activities of Type I wheat seedling nuclease was deter-
mined on an LKB 8101 electrofocusing column as descri-
bed in Methods. The result is diSplayed in Figure 6.
As expected, the two activities focused together and the

isoelectric point was determined to be 5.2.

Spectral Prgperties

The ultraviolet absorption Spectrum for the puri-
fied wheat seedling nuclease indicated an abnormal ratio
for the extinction indexes at 280 nm and 260 nm and was
therefore examined in some detail. The Azeo/Aago ratio
of the Type III purified enzyme after dialysis against
tris-HCl buffer at pH 8.0 varied between 1.09 and 1.30
depending on the individual preparation. This ratio
compares with 1.6-1.7 found for most proteins.

An abnormal distribution of aromatic amino acids

in the protein could account for the low Azao/Azeo

Figure 5:

45

Gel filtration of purified Type III

wheat seedling nuclease. Sephadex G-100
was packed in a column (1.9 x 80 cm) and
equilibrated with 0.1 mM Zn(Ac)2, 1.0

mM cysteine, and 0.05 M NaAc, pH 5.0.
Wheat seedling nuclease Type III was
applied as described in the text and

5 minute fractions were collected at a
flow rate of 0.4 ml/min. Every third

tube was assayed for protein by the tannic

acid method (absorbance at 650 nm),o

 

0.
Every second tube was assayed for DNase
activity (absorbance at 260 nm),0--——O.
Specific activity is plotted as DNase
activity (A250) per microgram of protein.
The arrows indicate positions where one
and two times the void volume of eluting

buffer had passed through the column.

.46

3

AEuagoN< .
:Eni esteem

 

M m.

 

Em can
2

 

*0 '3‘
...r m m
E.— onc .0 na< .,

     

3 - 6t 7
Frcclion Num er

a?

Table II

Molecular weight values and evidence
for Type III enzyme

of purity

 

 

Method Molecular weight

Evidence of purity

 

SDS gel electrophoresis 43.500
Sucrose gradient

sedimentation 45.000
Sephadex G-100 gel

filtration

Analytical ultra-
centrification 43.000

single major pro-
tein band

single symmetrical
peak

constant specific
activity across
elution peaka

single symmetrical
boundary

 

3See figure 5.

Figure 6:

48

Isoelectric focusing of DNase and 3'-
AMPase activities. Wheat seedling nuclea
Type I (800 units, 1 mg) was focused

on a LKB Model 8101 electrofocusing col-
umn as described in Methods. Fractions
of 1.5 ml were collected and enzyme assay
and pH were determined on every other

fraction. 3'-AMPase activity (absorbance

 

at 310 nm),a a; DNase activity

(absorbance at 260 nm),o

 

a: pH,0

 

86

0.

 

1

In al

I 260 um

 

49

 

 

Fraeflcn

 

 

 

Number

 

50

ratio observed for wheat seedling nuclease. To test this
possibility trytophan and tyrosine content in the Type
III enzyme was determined using the spectrophotometric
method of Edelhoch (48). A 0.18 mg/ml solution of Type
III enzyme was dissolved in 6 M guanidine hydrochloride
and the absorbance (measured on a Guilford spectrophoto-
meter in a 1 cm light path, 0.2 m1 cuvette) at 280 nm
and 288 nm was 0.130 and 0.095 respectively. Since the
molar extinctions for tyrosine and tryptophan are known
and the concentration of the protein in the cuvette can
be calculated (assuming a homogeneous protein), the two
unknowns, moles of tryptophan and moles of tyrosine per
mole of protein can be calculateduby solving two simul-
taneous equations. The result is 4.31 and 5.07 moles
of tryptophan and tyrosine per mole of wheat seedling
nuclease respectively.

Phenylalanine has an extinction maximum at 260
nm. The molar extinction coefficient at this wavelength,
however, is only about one tenth as large as tryptophan
and about one third as large as that for tyrosine at
280 nm. Therefore there would have to be a very large
number of phenylalanine residues per molecule of wheat
seedling nuclease to cause a low Azeo/Ageo ratio. The
spectra of phenylalanine has a characteristic fine struc-
ture region around 260 nm which produces multiple peaks.

For proteins with an appreciable amount of phenylalanine this

 

4

See appendix for calculations.,

51
fine structure causes irregularities in the protein Spec-
tra around this wavelength. Wheat seedling nuclease Type
III with an Aaeo/Aaao ratio of 1.26 was placed in a Cary
spectrophotometer and the spectra from 300 nm to 240 nm
was recorded. No obvious irregularities occurred in the
region around 260 nm indicating a relatively low content
of phenylalanine. It therefore seems likely that the
abnormal Azao/Aaeo ratio for wheat seedling nuclease
is not due to an abnormal distribution of aromatic amino
acid residues.

The final spectral properties studied were fluores-
cense excitation and emission Spectra. These Spectra,
along with UV absorption Spectra, were obtained with the
aid of a computer-centered Spectrophotometer-fluorimeter
constructed by Dr. J. Holland of this department. After
the appropriate spectra were obtained, the computer was
asked to construct a UV absorption Spectra which would
be produced if all the absorption at 285 nm,which had
produced fluorescence,were subtracted from the original
UV absorption Spectra. For a normal protein containing
only aromatic amino acid chromophores, this constructed
curve should be flat. The computer-constructed UV absorp-
tion Spectrum for wheat seedling nuclease, however, contained
a small peak at 255 nm. This result indicates the possible
presence of a chromophore present in wheat seedling nuclease

which absorbs light at 255 nm but does not produce any

52

fluorescence due to absorption at this wavelength.

Multiple Ionic Forms of Wheat Seedling Nuclease

During the initial attempts to develop a homogen-
eous purification of wheat seedling nuclease, polyacryl-
amide disc gel electrophoresis was used as a criterion
for estimating purity. It was noted that a number of
bands (6-12) persisted even when the specific activity
of the enzyme preparation had been considerably increased.

When wheat seedling nuclease Type III purified
enzyme (considered to be of high purity) is electrophor-
esed on polyacrylamide gels, 6-12 bands are easily dis-
cernible. Figure 7c Shows photographs of the multiple
bands produced by staining gels (for protein) containing
different percentages of acrylamide. The 7% acrylamide
gel was sliced lengthwise. One half was stained for pro-
tein while the other half was sliced into 2 mm Slices
and the DNase activity was eluted and assayed. The DNase
activity was associated with the third band. The first
band was independently determined to be a contaminating
protein from the PC purification step.

When proteins are electrophoresed on gels contain-
ing different percentages of acrylamide and the logarithm
of their relative mobilities on each gel are plotted
versus the percent acrylamide, the slope of the resulting
plot is a function only of the size of the protein and

the ordinate intercept is a function of the total

Figure 7a:

Figure 7b:

Figure 7c:

SDS gel electrophoresis of the last
three steps in the Type II purification
procedure. The amount of protein indi-
cated in the figure was layered on top
of SDS acrylamide gels and electrophoresed
as described in Methods. The arrow
indicates the length of the gels and

the direction of protein migration.
The-+ and —-signs indicate the polarity
of the electrodes during electrophoresis.
HS refers to the heat stable fraction,
CMC to the carboxymethyl cellulose frac-
tion and P-60 to the P-60 column step

of purification.

SDS gel electrophoresis of the last

two steps in the Type III purification
procedure. Notations are the same as

in figure 7a except PC refers to protein
from the phosPhocellulose step and

CMC refers to the final carboxymethyl
cellulose column of the Type III puri-
fication.

Polyacrylamide gel electrophoresis of
the purified wheat seedling nuclease
Type III on gels containing different
percentages of acrylamide. Electro-
phoresis was performed as described

in Methods except that it was terminated
before the tracking dye band was off

the gels. The 7% gel was sliced in

half lenghtwise and one half was stained
for protein while the other half was
sliced in 2 mm segments from which
enzyme activity was eluted. The arrow
indicates the direction of migration

of the protein bands which are numbered
chronologically from the top of the gel.

54

119m . . ,
Prozeln Sig: ion

16 CMC
10 P60

20 P60

 

 

‘ $1.0m“ ‘ g Italian

Ill-e e .0 [if
IIIIII I ea u) PC
a y 7.5 CMC
. is CMC

 

 

Parcaal Acrylamide
9

 

55

charge carried by the protein at the pH of electrophore-
sis (49). Using this procedure, wheat seedling nuclease
was electrophoresed on gels containing 5-9% acrylamide
(w/v). A plot of the logarithm of relative mobilities
for each band versus the percentage of acrylamide in the
gel is Shown in Figure 8. Band number one has a Slope
of 30 and was determined independently to be a contaminating
protein carried through from the PC purification step.
Using the same system, Hendrick and Smith (49) Showed
that a protein with a slope of 49 corresponds to a mole-
cular weight of 47,000. The plots in Figure 8 are all
approximately parallel, indicating that the differently
charged protein bands are all about the same size.
A slight increase in the Slope, 42-49, is noted as the
bands moved farther to the positive electrode. This
corresponds roughly to a difference of about 2,000 dal-
tons between bands 2 and 12. DNase activity was associa-
ted with only one band (number three) which represents
one of the least negatively charged proteins. Another
striking feature of the graph is that the vertical dis—
placement of the various plots is roughly the same bet-
ween any two bands indicating similar differences in
net protein charge between adjacent bands.

Isoelectric focusing of the purified Type III en-
zyme was performed in the hope of providing a technique
for studying the differently charged proteins. Figure

9 shows the results of focusing the purified enzyme on

56

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57

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a small, custom-made isoelectric focusing column. Most
of the protein formed a precipitant band which focused
at pH 2. A small amount of protein as well as all the
DNase activity focused at pH 5.2. The pH to which the
inactive protein focused is below the pK for any of the
commonly occuring amino acids.

In order to ascertain whether some or all of the
inactive protein bands found on acrylamide gels were
responsible for the overall low Aeso/Azeo ratio for the
Type III enzyme, gels were scanned at 260 nm and 280 nm
on a Gilford spectrophotometer equipped with a gel scan-
ning device. Figure 10 Shows the result of such a scan.
Only four major bands are easily discernible. Of these
bands, the Agao/AZGO ratio decreases as the bands become

more negatively charged.
Mechanism of Action During Enzymatic Catalysis

AS mentioned in the introduction to this thesis
a "mechanism of action" study is limited in descriptive
information by the amount and detail of the experimental
data available. This is eSpecially true for nucleases
where the substrates represent complex biomacromolecules.
The mechanistic areas of investigation reported here are
limited to those which illustrate unique features of the
enzymatic hydrolysis by wheat seedling nuclease as com-

pared with other nucleases appearing in the literature.

' —LL

Figure 9:

59

Isoelectric focusing of Type III enzyme.
The enzyme was focused in a small (154
mm x 10 mm) custom made column as des-
cribed in Methods. Fractions of 0.2

ml were removed and analyzed for DNase

 

activity (absorbance at 260 nm),0 a ,

protein (absorbance at 650 nm),o

 

.9

 

and pH,o c.

 

 

 

 

60

 

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Mlllllnelen Iron! Top 01 Gal

(3
The mechanisms involved in the hydrolysis of native, dou-
ble-stranded DNA molecules appear to have particular
Significance and are described in a separate section.
Exo-ang Endonucleolytic Cleavage

Early studies done in this laboratory by D. Hanson
(16) indicated that there may be a difference in the
mode of cleavage between dDNA and rRNA. Using the gel
filtration of reaction products after the method of Birn-
botm (50) it was Shown that dDNA is degraded by wheat
seedling nuclease primarily in an endonucleolytic fashion
and rRNA hydrolysis more nearly resembled the exonucleo-
lytic mode of cleavage. It was also observed (16) that
the presence of Zn4+ in the reaction mixture was slightly
inhibitory but increased the production of mononucleotides
during the hydrolysis of rRNA.

Early work in this investigation confirmed the above
results. When Zn" was included in the reaction with
rRNA as the substrate, the reaction products were even
more characteristic of an exoribonuclease when chromato-
graphed on Birnboim columns.

The Birnboim technique is a rough qualitative method
for these purposes. In order to define the mode of clea-
vage in a more quantitative manner the following experi-
ment was done. Polyadenylic acid was incubated at 37° C
with wheat seedling nuclease. The reaction contained

0.9 ml poly A (200 ODzeo units per ml), 8.0 ml 0.05 M

64
succinate buffer. pH 6 and 0.09 ml wheat seedling nuclease
(30 DNase units). At intervals of 10 minutes 0.9 m1
aliquots were removed and combined with an equal volume of
La(N03)3-HCl reagent. Acid-soluble material was determined
as described in Methods. Also at ten minute intervals 0.05
ml aliquots were removed and heated at 95° C for 3 minutes.
The amount of 5'-AMP in these samples was evaluated by
diluting the samples to 1.0 ml with 0.05 M succinate buffer.
pH 6 in a 1 cm light path cuvette. After the addition
of 5'-AMP amino hydrolase. the decrease in absorbance at
265 nm was measured in a Beckman DB spectrophotometer.
5'-AMP amino hydrolase deaminates only 5'-AMP (no deamina-
tion of oligonucleotides) converting it to 5'-IMP (38).
The difference in the molar extinctions between adenylic
acid and inosinic acid under these conditions is such
that a change of 1 OD at 265 nm equals the deamination
of 0.125 jimoles per m1 of 5'-AMP.

Figure 11 shows the results of the experiment des-
cribed above. The left hand ordinate indicates the amount
of 5'-AMP released and the amount of material (A260) con-
verted to acid-soluble form. The right hand ordinate is
calibrated as percent of 5'-AMP and acid-soluble material
produced during the reaction with 100% representing com-
plete degradation of poly A to mononucleotides. As can
be seen in the case of wheat seedling nuclease digestion
of poly A. the production of 5'-AMP and acid-soluble mater-

ial increases in a parallel fashion as would be expected

   

65

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canSHoalcho .o

 

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an pmoopoam anemones cansaonupaom .ellle «ommmaosc 93.3000
poems mo coauco on» an pmcopowm mz¢1.m arlllllaxaonucoc o no

can oum3 damage mam mcwxuoa acoauomum .uxmu as» aw canauonop

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cufi3.pocwnsoc cam cmuocapcw mass» on» an cc>osou 0H0; muoovaam HE
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66

alqnlos may spnpud go queued
o c c o

 

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7’. 7 ° 3

 

 

 

 

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30 40 50 60 70 80
Minnie: ol Incubation

20

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57

for an exoribonuclease. Theoretically the percentages

ch 5'-AMP and acid-soluble production should be equal
dxnring exonucleolytic degradation until the latter stages
of? the reaction. During the final stages of the reaction,
ESL-AMP production should continue after acid-soluble mat-
ezaial has ceased to increase since small oligonucleotides
arre acid-soluble. As can be noted from the figure, the
gu>int in the reaction which has produced 50% acid-soluble
products results in 18% production of 5'-AMP and similarly
2N1% acid-soluble products compares with 19% 5'-AMP. The
Slight lag in 5'-AMP production can be interpreted as a
Slight amount of endonucleolytic activity.

Figure 11 also shows the same experiment done with
quskmelon nuclease instead of wheat seedling nuclease.
BMuskmelon nuclease is known to be largely endonucleolytic
in its mode of cleavage of RNA (51). As expected for
a known endonuclease the mononucleotide production lags
substantially behind the formation of acid-soluble products.

The experiment in Figure 11 was repeated for wheat

4+

seedling nuclease with 1.0 mM Zn in the reaction mix-

4+

'ture. It was found that Zn had no effect on the shape

(of the curves or on the rate of the reaction.

2' Nucleotidase Activity
A number of different nucleotides were tested as
Substrates for the wheat seedling nuclease's monophospha-

tase activity and are compared in Table III. The reactions

68
Table III
DephOSphorylation rates on various nucleotide substrates

Rates were determined with the fixed-time phOSphate
assay described in Methods. The reaction mixture contained
0.05 ml of 50 mM dithiothreitol, 1 m1 of 2 mM nucleotide
(dissolved in 0.05 M sodium acetate, pH 5.0), 0.1 ml of
wheat seedling nuclease - Type III (diluted to contain
8 DNase units) and 1.35 ml of sodium acetate buffer (0.05 M,
PH 5.0 .

 

 

Substrate Ratea Percent of 3'-AMP rate
moles Pi/lS min %‘
}'-AMP 7.40 100
3'-UMP 3.60 46
j'-CMP 2.10 28
§'-GMP 1.68 23
2'-AMP 0.04 0
2'-UMP 1.94 27
2'-CMP 0.03 O
2'-GMP 0.04 0
3'-dAMP 3.04 41
3'-dTMP 0.51 7
3'-dCMP 0.03 0
3'-dGMP 0.05 1
5'-dXMP 0.05 1
5' -XMP 0.05 1

 

aRates listed are the average values of 2 determinations.
The background subtracted for the minus enzyme control was 0.05.

69
were carried out at pH 5.0 which is near the pH optimum
of the DNase activity. The 3'-ribonucleoside monophospha-
tes were the best substrates and were dephosphorylated
in the following order: 3'-AMP 7 3'-UMP ‘I 3'-CMP 7 3'-GMP.
Of the 3'-deoxyribonucleoside monophosphates only 3'-dAMP
and 3'-dTMP were dephosphorylated to any extent. These
two substrates were hydrolyzed at 41 and 7 percent of the
rate of 3'-AMP respectively. The only 2'-ribonucleoside
monophOSphate which was dephosphorylated was 2'-UMP at a
rate 27% that of 3'-AMP. This observed rate could be due
to the presence of some 3'-UMP in the commercial preparation
of 2'-UMP. None of the 5'-ribo- or deoxyribonucleoside
monophosphates served as substrates for the monophosphatase
activity. Thus, except for 2'-UMP, 3'-nucleotidase is
an appropriate classification for the monophOSphatase ac-
tivity of wheat seedling nuclease.

Figure 12 shows the pH profiles for the nucleoti-
dase activity on the four 3'-ribonucleoside monophOSphates.
The fixed time phosphate assay described in Methods was
performed at 0.3 pH intervals with the following buffers:
acetate-pH 4.0-5.2, maleate-pH 5.5-6.7 and tris-HCl-
pH 7.0-9.1. When 3'-AMP is used as a substrate, a rather
broad pH profile is noted. It is noted that the relative
rates of dephosphorylation for the various nucleotide
substrates changes with pH. The pH profile of nucleoti-
dase activity when 3'-GMP is used as a substrate shows

a fairly sharp optimum at pH 7.9.

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71

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72

Kinetic Analysis of thg_Hydrolysis of 3'-AMP and pApA

Kinetic analysis of the 3'-nuc1eotidase and RNase
activities was undertaken in order to study the relation-
ship of these activities in wheat seedling nuclease. A
continuous spectrophotometric assay was developed in order
to measure the initial rate of dephosphorylation of 3'-AMP.
This assay. which is described in Methods. measures the
decrease in absorbance at 265 nm produced when 3'-adenylic
acid is dephosphorylated to adenosine which is converted
to inosine by the coupling enzyme. adenosine deaminase.
Reaction mixtures contained 0.05 ml 0.2 M cacodylate buffer.
pH 6. 0.01-0.12 ml 3'-AMP (0.75 mM). 0-0.05 m1 inhibitor.
0.03 ml adenosine deaminase (1:500 dilution in 0.025 M
NHnAc. pH 8.0) and enough H20 to make 1.0 ml. The reaction
mixture was placed in a 1 cm light path cuvette and equili-
brated at 30° C in a water bath. It was then placed in
a Beckman DB spectrophotometer which had 30° C water
circulating through the cuvette chamber. The reaction
was initiated with the addition of 2.1 DNase units of
wheat seedling nuclease and the reaction rate monitored by
measuring the decrease in absorbance at 265 nm. Reaction
rates were linear for at least the first two minutes and were
proportional to enzyme concentration over a 10 fold range.

Figure 13a shows double reciprocal plots of substrate
concentration versus rate for the hydrolysis of 3'-AMP

with either no 5'-AMP present or 0.016 mm or 0.032 mm

Figure 15a:

Figure 13b:

73

Lineweaver-Burk plots for the hydro-
lysis of 3'-AMP by wheat seedling nuclease
Type II showing the inhibition by 5"AMP.
Reaction rates were determined at 30° C
using the continuous Spectrophotometric
assay for 3'-AMPase activity described
in Methods. Each reaction contained

0.05 ml 0.2 M cacodylate buffer, pH 6.0,
0.01-0.12 m1 3' AMP (0.75 mM), none,
0.025 or 0.05 ml 5' AMP (0.64 mM), 0.03
ml adenosine deaminase (diluted 1:500
with NH4Ac, 0.025 M pH 8.0) and enough
water to make 1.0 ml. The reaction was
initiated with 0.00215 ml of wheat seed-
ling nuclease Type II (2.2 DNase units).

0 . , 0.016 mM

 

e , no 5'-AMP;.

 

5'-AMP:I I, 0.032 mM 5'-AMP.

 

Lineweaver-Burk plots for the hydroly-
sis of 5'-AMP by wheat seedling nuclease
Type II showing the inhibition by 2',
3'-cAMP and ApA. The reaction conditions
were those described in the caption to
Figure 13a except 0.05 ml of 2',3'-cAMP
(0.82 mM) and ApA (0.75 mM) were used

instead of 5"AMP. e

 

e, no inhibitor;

 

 

A, 0.041 mM 2', 3'-cAMP; I g

0.038 mM ApA.

74

 

 

 

 

 

75
5'-AMP present in the reaction mixture. 5'-AMP is not
a substrate for wheat seedling nuclease or adenosine
deaminase. The inhibition produced by 5'—AMP is seen to
be competitive in nature. The Km and Vmax values for the
nucleotidase activity are 1.5 x 10.5 M and 1.3 x 102
‘llmoles/min/mg. The KI value for 5'-AMP inhibition is
calculated to be 1.1 x 10-5 M.

Figure 13b is a double reciprocal plot for hydro-
lysis of 3'-AMP in the presence of no inhibitor, 0.82 mM
2',3'-cyclic AMP, or 0.75 mM.ApA. The inhibition produced
by these compounds results in no change in the Km for the
reaction which is consistent with a noncompetitive type
of inhibition. Both of the inhibitory compounds of Figure
13b contain phosphodiester linkages. The KI values derived
from the inhibition by 2',3'-CAMP and ApA were calculated
to be 2.15 x 10-4 M and 1.2 x 10")4 M reSpectively.

When various salts were included in the reaction
mixture, inhibition of the rate of 3'-AMP hydrolysis was
observed. Figure 14a shows the result of making the reac-
tion mixture 0.05 M or 0.1 M in NaC1. The Lineweaver-Burk
plots reveal a competitive type of inhibition with a KI
for the salt-enzyme complex of 4.5 x 10”2 M.

In order to make kinetic measurements for the RNase
activity of wheat seedling nuclease an appropriate substrate
had to be found. When complex substrates such as nucleic

acid polymers are used, kinetic measurements are difficult

to obtain as well as to interpret. Therefore, a simple, well

Figure 14a:

Figure 14b:

76

Lineweaver-Burk plots for the hydro-
lysis of 3'-AMP by wheat seedling nu-
clease Type II showing the inhibition

by NaCl. The reaction conditions were
the same as those described in the cap-
tion to Figure 13a except either 0.05

ml or 0.10 ml NaCl (1 M) was used instead
of 5'-AMP. I

e, no NaCl;I I

 

 

8

 

0.05 M NaC1; I I , 0.1 M NaC1.

Lineweaver-Burk plots for the hydro-
lysis of pApA by wheat seedling nuclease
Type II showing the inhibition by NaCl.
The reaction rates at 30° C were measured
as described in the text. Each reaction
contained 0.05 ml succinate buffer

(0.5 M, pH 6.5) 0.1-0.4 m1 pApA (0.21
mM), 0-0.1 m1 NaCl (1 M), 0.005 ml

5'-AMP aminohydrolase and water to give

a total volume of 1.0 ml. Reactions were
initiated with 0.005 ml wheat seedling
nuclease Type II (1.2 DNase units)

0 , no NaC1; I

. ., 0905 M NaC1.:

 

 

 

I, 0.1 M NaC1.

77

 

 

 

”uncles/min

30

 

 

 

 

78
defined substrate was sought. The dinucleoside monophos-
phate ApA was not hydrolyzed by wheat seedling nuclease.
The dinucleoside diphosphate, pApA is a good substrate and
was made as described in Materials. Rate measurements
were obtained by monitoring the decrease in absorbance
at 275 nm produced by the cleavage of pApA yielding two
molecules of 5'-AMP which were converted to 5'-IMP by the
action of the coupling enzyme, 5'-AMP amino hydrolase.
This coupling enzyme, like adenosine deaminase, has a high
catalytic turnover number and is specific for 5'-AMP (ade-
nine, adenosine, ApA and pApA are not substrates), (38).

Saturating levels of substrate for pApA hydrolysis
by wheat seedling nuclease are relatively high. Therefore,
in order to measure rates at substrate concentrations
near the Km and still remain under an absorbancy of 2
in the Spectrophotometer,a wavelength was chosen where
the extinction of pApA would be lower than at 265 nm.

At 275 nm the molar absorptivity was considerably lower

and the difference in extinctions between 5'-AMP and 5'-IMP
at this wavelength was such that a difference of 1 0D
corresponded to the conversion of 0.43,;Lmoles/ml of

5'-AMP to 5'-IMP.

Figure 14b shows the Lineweaver-Burk plots for pApA
hydrolysis with and without NaCl present. In the absence
of NaCl the Km and Vmax are 9.1 x lO-SHand 1.67 x 103
‘kimoles/min/mg respectively. In the presence of NaCl

inhibition is seen to be noncompetitive in nature with the

79
K1 for the NaCl enzyme complex equal to 1.43 x 10.1 M.

To summarize these kinetic results, then, the non-
substrate, 5'-AMP, inhibits the nucleotidase activity
competitively while the phOSphodiester-containing non-
substrates, 2',3'-cyclic AMP and ApA, inhibit in a non-
competitive manner. Furthermore, NaCl acts as a noncom-
petitive inhibitor towards the phosphodiesterase activity
of wheat seedling nuclease using the RNA substrate, pApA,
while acting as a competitive inhibitor towards the 3'-
nucleotidase activity. These results are consistent with
the supposition that wheat seedling nuclease contains sep-
arate active sites (subsites); one site catalyzing the hydro-
lysis of the phosphomonoester bond of 3'-AMP and the other site

catalyzing the hydrolysis of the phosphodiester bond in pApA.

Base Preference at the Cleavage Site

Wheat seedling nuclease does not appear to possess
absolute base specificity towards single stranded substra-
tes; i.e., phosphodiester bonds adjacent to the four bases
are all hydrolyzed to some extent. Wheat seedling nuclease
does, however, have a preference for the base at the clea-
vage site. In other words, rates at the different sites
appear to vary considerably depending on the base present
next to the bond being cleaved. For example, if the mono-
nucleotides produced during a digestion of dDNA by wheat
seedling nuclease are separated from the other reaction

products, the relative content of 5'-dAMP, 5'-dTMP, 5'-CMP

80
and 5'-dGMP is 100:}8:l6:8 (16).

As has already been noted from the results in Table
III, the rate of dephOSphorylation of the various 3F-nu-
cleotides varies in the following order 3'-AMP3'3'-UMP'7
(3'-‘TMP) >3'-CMP>3'-GMP. In this case the specificity
of the 5'-deoxyribonuc1eoside monophosphates is even more
pronounced since 3'-dCMP and 5'4iGMP are not even hydro-
lyzed at detectable rates.

The same relative order of base preference is
observed when the ribohomopolymers are digested with wheat
seedling nuclease. Rates of hydrolysis of the various
ribohomopolymers were measured at 306 C using the hyper-
chromic assay described in Methods. The reaction mixture
was 0.05 M in acetate, pH 5.0 and contained 0.7 ODZQO
units of each of the polymers. The initial rates of hydro-
lysis of poly U, poly C and poly G were 53%, 10% and 4%
respectively as fast as the rate of poly A hydrolysis
by wheat seedling nuclease. It should be pointed out,
however, that the amount of secondary structure of the
polymers varies considerably under the reaction conditions.
Poly G, for example, possesses considerable secondary
structure.

The results of the base preference experiments are
summarized in Table IV. As can be noted, the same rela-
tive order of base preference is present in all of the
experiments.

The interaction of poly G with wheat seedling nuclease

1
8

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'82
was investigated in more detail. Acid-soluble assays
were carried out as described for the DNase assay except
poly G was used instead of dDNA. No detectable formation
of acid-soluble material occurred even after 30 minutes
with 16 DNase units of wheat seedling nuclease. This
represents ten fold more enzyme than would be needed to
degrade dDNA completely to acid-soluble material in 30
minutes. Likewise no formation of acid-soluble material
occurred when the reaction was performed at pH 7.5.

Poly G was found to be an excellent inhibitor of
the DNase activity. When 0.03 mg/ml of poly G was included
in the standard DNase assay, 50% inhibition resulted.

Poly G was also studied as an inhibitor of the 3'-
nucleotidase activity. Using the kinetic system outlined
in the previous section, Lineweaver-Burk plots were obtain—
ed for 3'-AMP hydrolysis with and without poly G present
in the reaction mixture. The rates of the reactions with
poly G present produced non linear, concave upward double
reciprocal plots indicating a complex 'inhibitor-enzyme

interaction.

Mechanism of Action on Native DNA

It had been noted early in the investigation of
wheat seedling nuclease that single-stranded, denatured
DNA was a much better substrate than native, double-strand-

ed DNA. In fact, the rate of formation of acid-soluble

83
products from native DNA (nDNA) was only a few percent
that released from denatured DNA (dDNA) using commercial
preparations of salmon sperm and calf thymus DNA. There
were several possibilities which might explain the low
rate with nDNA. First. the small amount of acid-soluble
material released from native DNA could arise from the
hydrolysis of small amounts of dDNA or RNA which were endo-
genous to the commercial DNA preparation. Secondly. a
contamination of another nuclease in the wheat seedling
nuclease preparation might be responsible for the action
on nDNA. A third possibility remained that wheat seedling
nuclease possessed the ability to hydrolyze nDNA to a
limited extent. In order to investigate these possibilities.
a study was conducted on the hydrolysis of native DNA
with a highly purified preparation of wheat seedling

nuclease.

Hydrolysis of Double-Styand Sygyhetic Pglyggys
The alternating copolymeric duplex. poly d(ApT).

was compared as a substrate to native and denatured calf
thymus DNA using the hyperchromic assay described in
Methods. Reactions were carried out at 30° C in 0.04

M cacodylate. pH 5.0. Under these conditions poly d(A-T)
was hydrolyzed at a rate comparable to dDNA as can be seen
in Figure 15. Under these same conditions nDNA. and the
synthetic duplex. poly dG-poly dc. showed only a

very slight increase in absorbance at 260 nm.

84

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86

The effects of ionic strength and pH on the hydro-
lysis of poly d(A-T) were looked at in some detail. It
was found that the rate of hydrolysis of poly d(A-T) by
wheat seedling nuclease increased sharply as the pH was
lowered from 7 to 4.5, especially at low salt concentra-
tions. For example, at pH 4.8 in 0.04 M cacodylate
buffer, poly d(A-T) was hydrolyzed about 10% faster than
dDNA as estimated by the hyperchromic assay. Figure 16
shows the effect of pH and ionic strength on the hydrolysis
of poly d(A-T). At pH 5.0 in 0.2 M cacodylate, the rate
for poly d(A-T) was only 30% that of dDNA. When the
reaction was carried out at pH 6.2 the rates for poly
d(A-T) at 0.04 M, 0.1 M and 0.2 M cacodylate were 18%,

8% and 0% respectively when compared to the rate of dDNA
hydrolysis. The melting transition temperatures, Tm's,
for poly d(A-T) were measured under the various assay
conditions and found to be at least 20° C above the 30° C
temperature used in the assays.

Figure 17 shows the time course for the hydrolysis
of poly rA,U, native gh-l DNA and denatured gh-l DNA. In
this case the DNA substrate was uniformly labeled with
32F and the RNA synthetic polymer was 3H labeled. Acid-
soluble assays were performed as described in the figure
caption. As can be seen from the figure, poly rA,U and
denatured gh-l DNA were rapidly converted to acid-soluble
form whereas native gh-l DNA showed no decrease in acid-

insoluble material over a period of one hour.

87

Figure 16: Ionic strength and pH effects on the

initial velocities of hydrolysis of poly
d(A-T) and dDNA. Poly d(A-T) and dena-
tured calf thymus DNA (0.5 ODzeo units)
were dissolved in 1 m1 cacodylate buffer
and initial rates were measured at 37°

C using the hyperchromic assay described
in Methods. Cacodylate buffers were pre-
pared by dissolving the indicated concen-
tration of cacodylate in water and ti-

trating to the indicated pH with NaOH.

 

 

 

I e , dDNA in .04 M buffer;I I
dDNA in 0.1 M buffer; I IdDNA in
0.2 M buffer; o--—&o , poly d(A-T) in

0.04 M buffer;a.—_—c , poly d(A-T) in
0.1 M,buffer;o—n—-o, poly d(A-T) in

0.2 M buffer.

 

88

A.

o—

w :.E\Ecoou .0 na<<v

 

Figure 17:

89

Time course for the loss of acid insol-
uble substrate during the hydrolysis

of native gh-l DNA, denatured gh-l DNA
and poly rA,U. The reaction with gh-l
DNA contained 0.150 ml 32? gh-l DNA
(550 ,qgms/ml, 131 cpm/Agms), 0.06

ml 0.5 M acetate buffer, 0.06 ml 10 mM
2-mercaptoethanol, 0.24 ml H20 and 0.09
ml Type II enzyme (3 DNase units). The
reaction was carried out at 30° C and
0.09 ml aliquots were removed at the
times indicated and combined with 5 ml
cold TCA. Salmon sperm carrier DNA was
added and the precipitate was collected
and washed on a filter, dried and count-
ed in scintillation fluid. The denat-
ured DNA was made by heating the gh-l
DNA at 100° c for 10 minutes followed
by quickly cooling in ice. The reac-
tion with rA,U’was done the same way
except that it contained 0.025 ml poly
r(A,U) (2 mg/ml and an labeled at 3820
cpm per ,UJ).I-——————I, native gh-l

DNA;O

 

I , denatured gh-l DNA;o---o,

poly rA,U.

90

20

6
I

Ambhw Envy In
8

4

 

 

1

' '8'--1\

4 0 5 0 6 O
lncubciien

3O

20
Minutes of

10

91

.gimit Digests of Nativewggg

The action of wheat seedling nuclease on naturally
occurring native DNA molecules was investigated next.
Commercially available preparations of DNA were not satis-
factory for this purpose due to a) possible contamination
with denatured DNA, RNA or enzymes and b) the heterogen-
eity produced by random shearing of the DNA during pre-
paration, shipment and storage. Either high molecular
weight g, 231; DNA or intact viral DNA molecules were
used in the following studies.

The first substrate to be tested was g, 921; DNA.
A high molecular weight preparation was made as described
in Materials. The action of wheat seedling nuclease on
native g, gglg.DNA was monitored by measuring viscosity
changes during the time course of the reaction. In order
to correlate viscosity with molecular weight of the DNA,
an ultra low shear viscometer capable of measuring small
volumes of DNA was used.5 The reaction (2.0 ml) was con-

ducted at 22° C at pH 5.0, and contained 39.4,xAgms E, coli

 

DNA, 0.04 mmoles acetate buffer, 0.002 mmoles 2-mercapto-
ethanol, 0.38 mmoles NaCl. The reaction was initiated

by adding 10.5 DNase units of wheat seedling nuclease

Type II and 0.35 ml aliquots were placed in the visco-
meter at the times indicated. Viscosity measurements

were obtained and molecular weights calculated as described

in Methods.

 

5These experiments were conducted at the veterans Admin-
istration Hospital, Research Division, Bedford, Massachusetts.

92

The time course for this reaction is plotted in
Figure 18. The striking feature of this plot is the
appearance of a limit DNA polymer (MW='2.6 x 10°) which
is resistant to further hydrolysis for extended periods
of time even after additional enzyme is added. The de-
crease in molecular weight with time appeared to be an
exponential function. When the limit molecular weight
was subtracted from the molecular weight of the reaction
products during the first 4.5 hours and the logarithms
of these molecular weights were plotted versus the time
of incubation, a straight line resulted (inset to the
figure). The first order kinetics observed suggest that
all the cleavage sites are hydrolyzed at approximately
equal rates by wheat seedling nuclease.

When the reaction was conducted at pH 6.4 a decrease
in molecular weight of about 10 x 106 was observed during
the first hour. After that the molecular weight remained
at 60 x 106 even after additional enzyme (10.5 units)
was added at 4 hours. At pH 8.0 no reduction in mole-
cular weight was observed during the first two hours of
incubation.

The interaction of intact linear duplex viral DNA
molecules with wheat seedling nuclease was studied next.
Figure 19 shows the result of incubating ADNA with wheat
seedling nuclease followed by sedimenting the reaction
products on an alkaline sucrose gradient. Reaction mix-

tures of 0.1 ml containing 0.03 mg A DNA, 5 limoles acetate

Figure 18:

93

Time course for the decrease in mole-

cular weight during the hydrolysis of
native E, £211 DNA by wheat seedling
nuclease Type II. Reaction mixture con-
tained 0.08 ml 0.5 M sodium acetate

buffer (pH 5.0), 0.02 ml 0.1 M 2-mercap—
toethanol and 1.92 ml E, £21; DNA
(20.5,xxgms/m1 in 0.2 M NaC1) p1us 10.5
DNase units of enzyme. The reaction

was carried out at 22° C and an additional
10.5 units of enzyme were added at 4 hours.
At the times indicated, 0.35 ml aliquots
were removed and placed in the visco-

meter and Viscosities and molecular weights
were determined as described in Methods.
The inset in the figure is a replot of

the same data after subtracting the "limit"
molecular weight of the reaction products
and taking the logarithm at the various

stages of hydrolysis.

94

1o
3;
60 : 7.6
"2
‘T
5 3
‘2. 3.
'5 o as
.. I . o
f. " o
'3
3
..3
.2 6'0 1 2 3 4
3 Hours
_’
°20
1 - 10.5
Unlfs o! 7
1o Emmi 21.6110:
I--0
1 2 3 4 5 6 11

Hours oi lncuballon

95

buffer, 0.1 ,umole 2-mercaptoethanol and 1.0 DNase unit
wheat seedling nuclease Type II were incubated at 22° C
at pH 5.0. The reactions were terminated at the times
indicated by the addition of 0.015 ml 1 N NaOH, 0.03 ml
4-5 M NaCl and 0.005 ml H20. After standing for half
an hour at room temperature, 0.1 m1 samples were layered
on 5-20% alkaline sucrose gradients and centrifuged as
described in Methods. The molecular weight of the DNA
from the 12 hour digest was calculated to be 1.05 x 10°.
This value corresponds to 2.09 x 10° for the weight of
the equivalent duplex form. After 2 hours of incubation
61 large percentage of the reaction products were about
llalf the original size of ADNA, suggesting many early
(:leavages near the middle of the ADNA molecule. It is
interesting to note that ADNA possesses a well characterized
Ix-T rich region near its center.

When ADNA was incubated under conditions described
above for 24 hours with an additional 1.0 DNase unit of
enzyme added at 18 hours, the molecular weight of the
reaction products were still approximately 1 x 10°.
When a 24 hour digest of ADNA was sedimented in a neutral
Sucrose gradient, the resulting molecular weight of the
diigestion products was calculated to be about 2 x 10°.
Since DNA exists entirely as single-stranded molecules
during alkaline sucrose gradient sediemntation, the above
result indicates that wheat seedling nuclease produces

exclusively double—strand scissions in ADNA.

96

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canoe moasomaoe 2 meme none; :oeuemod was .mooeumz ca owneuo
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nos mcaaomwm umon3_HE H0.0 new owm HE H0.0 .Hoaonuooummoamslm
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onv mza A as 00.0 cmaflmucou mousuxae coauommm .muoowoum soap

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98

Gh-l DNA is a linear duplex DNA (m = 23 x 106)
containing 57% G + c and 43;: A + T (52). A limit digest
averaging 3.0 x 106 daltons is obtained as indicated by
the results of sedimentation in neutral sucrose gradients
which are summarized in Figure 20. Additional incubation
for 24 hours with an extra 1.0 DNase unit of enzyme added
at 18 hours results in no further reduction in molecule size.

Electrophoresis of the limit polymers produced from
gh-l DNA was performed with agarose gels as described
in Methods. using fi-Xl74RF DNA as a 3 x 106 dalton marker.
The limit polymers migrated as a single wide band. the
center of which corresponded to the distance moved by the
marker. Greater than 90% of the material in the limit
polymer band was Judged to be within 2 0.5 x 106 daltons
of the marker (MW = 3.0 x 106).

Table V compares the molecular weights found for
various DNA's after hydrolysis with wheat seedling nuclease
for 24 hours as described above for A.and gh-l DNA. T4
DNA produced the smallest limit polymers (1.8 x 106 daltons -
duplex equivalent molecule weight). The polymers produced
by extensive treatment of gh-l DNA with wheat seedling
nuclease yielded the largest products (3.0 x 106 daltons).

Mechanism o; gh-l DNA gygzglysis
DNA from the bacteriophage gh-l was chosen as a

Figure 20:

99

Neutral sucrose gradient sedimentation

of gh-l DNA and its reaction products.

The reaction mixture contained .05 ml

32F gh-l DNA (445 lxgms/ml, 117 cpmAugms),
0.015 ml 10 mM 2-mercaptoethanol, 0.045
ml H20 and 0.025 ml Type II enzyme (0.5
DNase units). The reaction was conducted
at 22° C and 0.1 m1 aliquots were removed
at the times indicated and combined with
0.025 ml 1.5 M tris base. Neutral sucrose
gradient sedimentation was accomplished
as described in Methods. The 8 value

and molecular weight calibration shown

on the figure is based on the sedimenta-
tion of gh-l DNA as a marker (31 S,

23 x 106 daltons).

100

S 31 25;? I; 195 4 31 25201; 105
Man" 3; 11; 5.8 #21 q; 11.; 5|; 1:“
a

 

       
       

6 o No Enzyme 6 4.5 Hrs
s 1\. i
:4 / :4 MN
::2/0 :2 ,1. o
a 4. 8 or ‘g‘

q'°°‘°°°'¢eooo-o°oc°'°°°°

    

    
 

/o Sucrose

0’“: 3 H I'S

32F (cs-11:10")

101

Table V

Summary of molecular weights of limit digest

polymers from various DNA sources

 

 

 

 

 

DNA Molecular weight of limit polymer
molecular single double

source weight strand method strand method
. 6 6

E. coli 75 x 10 2.6 x 10 c

6

T7 24 x 106 1.2 x 10 a

T4 130 x 106 0.9 x 106 a

A 31 x 106 1.05x 106 a 2.0 x 106 d

gh-l 23 x 106 1.7 x 106 a 3.0 x 106 d

1.49x 106 b

3 1 x 106 e

 

a Alkaline sucrose gradient sedimentation

b.32P end labeling (see Methods and Table VII)

c Viscometric analysis
d Neutral sucrose gradient sedimentation

e Agarose gel electrophoresis

102

substrate for a more detailed study of the action of
wheat seedling nuclease on native DNA.

. The production of limit polymers from various intact
DNA molecules suggested that only a few specific sites
in the DNA molecule were being cleaved. Earlier studies
with commercial preparations of DNA indicated that a
small amount of acid soluble material (up to several per-
cent as much as that liberated from dDNA) was produced
during hydrolysis. This acid-soluble production from
commercial DNA's however could arise from a) dDNA, RNA, or
enzyme contamination or b) heterogeneous ends of sheared
DNA as was pointed out earlier. The viral DNA's used
were essentially free of these complications. RNA is not
present in the phages and the DNA was shown to have mole-
cular weights equal to the known values for intact DNA
in alkaline or neutral sucrose gradients. Therefore this
DNA is native, intact and unnicked.

Acid Soluble Assays Acid soluble assays were per-
formed on uniformly labeled 32F gh-l DNA. Initial studies
with Type II enzyme indicated that several percent
of the total 32p was converted to acid-soluble material
during extensive hydrolysis of gh-l DNA. This activity
was eliminated,however,by adding the P-60 chromatography
step to the Type II purification (see "Purification"
section of Results). Table VI lists the percentage of
32F distributed between acid-soluble and acid-insolub1e

products after extensive hydrolysis of 32 P gh-l DNA at

103

Table VI

Acid soluble material produced during digestion
of 32? uniformly labeled gh-l DNA by wheat seed-
ling nuclease at 22° and 37° C.

Reaction mixtures contained 0.1 ml 32P gh-l DNA (15
.ugms, 3 x 103 cpmnugm) 0.03 ml sodium acetate buffer (0.5
M, pH 5) 0.003 ml 100 mM 2-mercaptoethanol, 0.159 ml H20
and 0.008 ml Type-II enzyme containing 0.5 DNase units.
Reactions were terminated by adding 0.2 m1 salmon sperm
DNA (1 mg/ml) and 0.5 ml cold La(N03)3 - HCl reagent.
After standing for 20 minutes at 4° C the samples were
centrifuged at 10,000 x g for 20 minutes. Cerenkov radia-
tion of the acid soluble fraction was counted directly and
the acid precipitate was dissolved in 0.9 m1 H20 plus 0.1
m1 1 N NaOH, After counting, 0.1 m1 of the redissolved
acid precipitated fraction was sedimented in an alkaline
sucrose gradient for evaluation of the molecular weight
as described in Methods.

 

 

 

Molecular
Acid Acid weight of
soluble precipated digestion
Temp. Time material material products
guC hours cpm %* cpm 7t—
220 18 14 0.04 43,512 99.96 3.2 x 106
37° 3 29 0.07 ' 41,822 99.93 2.7 x 106

 

amolecular weights determined on single strand polymers,
but have been multiplied by 2 and reported as duplex equi-
valent values

104

22° c and 37° c. Sucrose gradient analysis indicated
that the products had been reduced to ”limit“ size during
the incubation with wheat seedling nuclease. All of the
32? radioactivity in the starting material was recovered
as acid-soluble or acid-insoluble material. Only 14 and
29 counts per minute of 32F radioactivity were present
in the acid-soluble fraction. These values were based
on ten minute counts after subtracting 30 cpm due to back-
ground radiation. The background radiation was found to
vary between 24 and 40 cpm. Thus the 32F in the acid-soluble
fraction represents a negligible amount of radioactivity.

Izanscyiptign of gh-l DNA Limig gnlymeyg Since the
size of the average transcriptional unit in gh-l DNA is
thought to be about 3 x 106 daltonsé. the possibility
existed that the limit polymers produced by extensive
hydrolysis with wheat seedling nuclease were separate trans-
criptional units. Accordingly. they were examined as temp-
lates for the transcribing activity of gh-l phage-induced
DNA-dependent RNA polymerase. Limit polymers of gh-l DNA
hydrolysis were sedimented on a neutral sucrose gradient
and the fractions from the gradient were assayed for DNA
and polymerase template activity. .As can be seen from
Figure 21. DNA. throughout the peak served as an acceptable
template for transcription.

Material from the three peak fractions (0D ) of

260
the experiment shown in Figure 21 was pooled and dialyzed

against 10 mM tris-301. pH 8 and 0.05 M NaCl. These

6Personal communication from Dr. J. Boezi.

 

Figure 21:

105

RNA polymerase activity on a limit di-
gest of gh-l DNA after sedimentation
through a neutral sucrose gradient.

The limit gh-l DNA digest was produced

by the following reaction: 0.5 m1 gh-l
DNA (680 ,Ugms/ml), 1.0 ml 0.1 M acetate
buffer, pH 5.0, 0.02 ml 100 mM 2-mercap-
toethanol, 0.38 ml H20 and 10 DNase units
of Type II enzyme. The reaction was
carried out at 22° C for 20 hours with

an additional 10 units of enzyme added

at 16 hours. The reaction was terminated
with the addition of 0.4 m1 of 1.0 M

tris base. A 0.1 m1 aliquot was sedi-
mented through a neutral sucrose grad-
ient as described in Methods. Aliquots
of 5 )ul from every second fraction were
used as substrate for the gh-l phage-
induced DNA dependent RNA polymerase
reaction described in Methods. Molecular
weights shown in the figure were calcu-

lated using gh-l DNA as a marker (31 S,

 

23 x 10" daltons). o e , gh-l DNA
limit polymer (absorbance at 260 nm);

 

a, RNA polymerase activity (SH-CTP

incorporated).

Abs II 260!!!“

106

uw=3x1o -
t

I"

haw-115x10"

 

% Sucrose

3H (cpm x 10'3)

107

polymers (gh-l DNA pieces). as well as whole gh-l DNA
molecules. were used as substrates to study the kinetics of
the transcription reaction. Figure 22 shows the substrate-
saturation curves as well as the double reciprocal sub-
strate versus rate plots when these two templates were
used. when the gh-l DNA pieces were used as a template
the Km - 2.78 x 10'6 M and the Vmax 8 l x 103 units/mg.
0n the other band. using whole gh-l DNA molecules as temp-
lates. the Km = 5.56 x 10-6 M and the Vmax - 3.17 x 103
units/mg. Thus the maximum rate of transcription for the
gh-l DNA pieces is almost one third that for whole gh-l
DNA molecules.

Bu an s t n - t e s

Given the fact that gh-l DNA is 23 x 106 daltons and the

limit polymers are all approximately 3 x 106 daltons. an
average of about 7-8 DNA pieces are produced from each
gh-l DNA molecule by the action of wheat seedling nuclease.
In order to ascertain whether any of these DNA pieces
contained abnormal guanylic p1us cytidylic acid content.
0301 gradients were prepared and the DNA was handed during
centrifugation as described in Methods. The results are
shown in Figure 23. The DNA pieces all banded at fractions
with the same refractive index (and therefore at the same
buoyant density) as the whole gh-l DNA molecules. Thus
the average 01+ 0 content of the limit polymers were all
about the same as that of the whole gh-l DNA:Iolecule

(57X 6" C).

Figure 22a:

Figure 22b:

108

Substrate saturation and Lineweaver-
Burk plots for the transcription of
gh—l DNA by gh-l phage-induced DNA
dependent RNA polymerase. Assays

for polymerase activity were conducted
as described in Methods except that
reaction volumes were 0.5 m1 and 0.125 ml
aliquots were withdrawn and assayed

at 5 minute intervals. In all

cases reaction rates were linear

for the first 15 minutes. One

unit of polymerase activity

equals 1 nmole 3H-CTP made acid
insoluble per 10 minutes. The
concentration of DNA is reported

as the molarity of nucleotide
equivalents and determined by
measuring absorbance at 260 nm

(1 mEll/ml ‘20 ODzeo) .

Substrate saturation and Lineweaver-
Burk plots for the transcription of
gh-l DNA limit polymers by gh-l
phage induced DNA dependent RNA
polymerase. Limit polymers of gh-l
DNA produced by the action of wheat
seedling nuclease (see Figure 21)
were sedimented on neutral sucrose
gradients and the fractions cor-
responding to material with a mole-
cular weight of 3 x 106 were pooled
and dialyzed against 10 mM tris-HCl,
pH 8.0 and 0.05 M NaC1. Using this
material as substrate, kinetic mea-
surements were made as described in
Figure 22a.

Enzyme Activity (U nits)

Enzyme, Activity (Units)

109

 

[9MBDNA Plec6§ (“"0636

 

Figure 23a:

Figure 23b:

110

Buoyant density bending of gh-l DNA
in a CsCl gradient. 32 P labeled gh-l
DNA (30llgms) was layered on top of a
CsCl solution and banded as described
in Methods.

Buoyant density banding of gh-l DNA limit
polymers in a CsCl gradient. The limit
polymers were made as described in Figure 21.
After layering 30Mgms on top of a CeCl
solution. banding was accomplished as des-
cribed in Methods.

32? (cpm x 104)

lbs at 260nm

111

    

16 24 32
Fraction Number

 

1.3980

o
§
0

Retractive Index

  

4040

Rail-active Index

112

Nucleotide composition at 3} end ofggh-l DNA limit
polymers In order to determine the nucleotide composition
near the cleavage site in gh-l DNA, a small number of
nucleotides were cleaved from the 3' termini of the gh-l
DNA limit polymers by the action of venom phosphodiester-
ase. The reaction contained 0.1 ml 32P gh-l limit polymers
(20,/ugms/ml, 1.05 x 10‘ cpm/Jugms), 0.06 ml 0.7 M glycine
buffer, pH 9.2, 0.03 ml 0.3 M.MgCla and 0.11 ml H20.
Venom phosphodiesterase was added (0.01 ml which had pre-
viously been treated to remove trace amounts of 5i-AMPase
activity as described in Methods) and the mixture was in-
cubated at 220 C for 15 minutes. The reaction was term-
inated by adding 0.2 ml salmon sperm DNA (1 mg/ml) and
0.5 ml cold 10% TCA. After standing in ice for 20 minutes
the precipitate was collected by centrifugation at 20,000
x g for 20 minutes. The supernatant was counted directly
as Cerenkov radiation and the precipitate was redissolved
in 0.01 N NaOH and counted. The acid-soluble portion con-
tained 584 cpm of 32F and the acid-insoluble portion con-
tained 20,752 cpm. This represents the conversion of
2.6% of the gh-l DNA limit polymer to acid-soluble material.
The limit gh—l DNA polymers have an average molecular
weight of 3 x 10°. This corresponds to 9.1 x 103 nucleo-
tides per limit polymer or a chain length of 4.55 x 103
nucleotide pairs. venom phosphodiesterase removes nucleo-
tides progressively from the 3' terminus of DNA. Therefore
2.6% conversion of the gh-l DNA limit polymers to acid-

soluble material represents the cleavage of 118 nucleo-

113
tides on the average from both 3' termini. To these nucleo-
tides contained in the acid soluble supernatant was added
0.2 ml 0.7 M glycine buffer, pH 9.2 and the mixture was
titrated to pH 9 with l N NaOH. The four deoxynucleotides
(0.05 ml each 10 mg/ml pdc, pdA, pdG, pdc) were added
and the nucleotides were separated on a Dowex column as
described in Methods. Table VII compares the 8"’P radio-
activity eluting in each nucleotide peak on a percentage
basis. The nucleotides released from the exonuclease action
are 46% A+ T and 54% G+C. For comparison the same experi-
ment was done on whole gh-l DNA. In this case the GtC
mole percent equals 57% and Ai-T equals 43% which agrees
with that determined by other methods for gh-l DNA (52).
Thus the area representing an average of 118 nucleotides
from the cleavage site is not greatly different in nucleo-
tide composition as compared to the entire gh-l DNA mole-
cule although a slight enrichment of Ai-T‘was found.

322 5' End Labeling of gh-l DNA Limit polymers The
production of limit polymers from native DNA by wheat
seedling nuclease yielding less than 0.1% acid soluble
material suggests a specific, well defined site of cleav-
age on the DNA molecule. In order to learn more about
this specificity, polynucleotide kinase was used to label
the 5' termini of the limit gh-l DNA polymers with 32?.
Polynucleotide kinase catalyzes the reaction which trans-
fers the 3 phosphate of ATP to the 5' OH and of a poly-

nucleotide chain. Thus by using ATP labeled in the X

Nucleotide composition at the 3' end of uniformly
32P labeled gh-l DNA limit polymers

114

Table VII

Acid soluble material representing 2.6% of the total

32P counts in the limit polymers was produced by the limited

action of venom phosphodiesterase (see text for details).
This material containing the nucleotides released by the

venom exonuclease was applied to a dowex column and the
nucleotides were separated as described in Methods.
comparison, uniformly 32F labeled gh-l DNA was reduced

For

completely to nucleotides by the combined action of DNase
I and venom phosphodiesterase and the individual nucleo-
tides separated as described in Methods.

 

 

 

Nucleotide Acid soluble fraction gh-l DNA

cpm _—7%7 cpm 5%
P40 123 27 835 27
Pdl 123 26 614 20
pdG 127 27 941 30
pd? 98 20 751 23
pdA.- pdT 46 43
PdG 3 PdC 54 57

 

115
position with 32F it is possible to label the 5' terminus
of a polynucleotide chain,providing there is a 5' OH group
present at the terminus.

The limit gh-l DNA polymers were made in the follow-
ing manner. A reaction mixture of 1.0 ml at pH 5 contain-
ing gh-l DNA (354 1agms), l ,umole 2-mercaptoethanol, 0.05
mmoles acetate buffer and 10 DNase units of wheat seedling
nuclease was incubated at 220 C for 18 hours. An additional
10 units of enzyme were added and the incubation was con-
tinued for 2 more hours. SDS (0.05 ml, 10%) was added
and the solution was incubated at 60° C for 5 minutes.
After two phenol extractions, the aqueous phase was dia-
lyzed for 3 days against 3 changes of 50 mM KCl and 0.01
M tris-HC1, pH 8.0.

The limit polymers as well as whole gh-l DNA mole-
cules were labeled with 32F by the following procedure.

DNA (0.1 m1 295 ,ugms/ml gh-l DNA or 0.1 m1 280 ,ugms/ml
gh-l DNA limit polymer) was denatured by adding
0.11 ml 1.0 M NaOH and the solutions were kept at room
temperature for 5 minutes. Next, 0.11 ml of 1.0 M tris-
HCl, pH 7.8 was added. After adding 0.005 ml alkaline phos-
phatase (containing 0.037 units and prepared as described

in Methods) the reaction was allowed to proceed at 37°

C for 30 minutes. An additional 0.005 ml of alkaline phos-
phatase was added and the incubation continued for 30 min-
utes. This procedure was necessary to remove the phosphate

group at the 5' terminus of the limit polymer leaving a

116

5' on group to react with BZP-ATP. After adding 0.28 ml
820 the reaction mixtures were phenol-extracted twice with
equal volumes of water-saturated. freshly distilled phenol.
The aqueous phases were then dialyzed for 48 hours against
4 changes of 0.01 M MgCl2 and 0.07 M tris-801. pH 7.6. To
each sample was then added 0.05 ml 50 mM dithiothreitol and
0.01 ml 32PnATl" (l.27pmoles/al. 2.38 x 108 cpm/,umole).
The reaction was started by adding 0.02 ml of polynucleotide
kinase (5 units). The reaction mixtures were incubated at
37° c for 90 minutes with an additional 0.02 ml of poly-
nucleotide kinase added at 30 and 60 minutes. The samples
were then dialyzed against 0.5 M NICl and 0.03 M tris-801.
pH 7.6 for 48 hours with 3 changes of buffer. They were
then dialyzed against 2 changes of 0.01 M MgClz and 0.02 M
tris-HCl. pH 7.6. over a 24 hour period. At this point all
of the 32? had been dialyzed away from the samples which
had no polynucleotide kinase treatment.

The samples were removed from.the dialysis tubing and
counted directly by measuring the Cerenkov radiation.
The results are shown in Table VIII. The controls lacking
phosphatase treatment indicate that the gh-l BIA contained a
significant number of free 5'-OR groups. a number not increased
by prior treatment with alkaline phosphatase. However. treat-
ment of the limit polymers with phosphatase markedly increased
the number of 5'-OH groups capable of undergoing the polynu-
cleotide kinase reaction. The high value encountered in the
case of the minus-phosphatase control may reflect the presence

of some phosphatase activity during the kinase reaction.

117

Table VIII

32F incorporation into gh-l DNA and gh-l DNA limit
polymers by polynucleotide kinase

The method used for end labeling with polynucleotide
kinase is described in detail in the text. All samples
were carried through the alkaline phosphatase and poly-
nucleotide kinase treatments except the Specified enzyme
was left out where noted. The final reaction mixture was
dialyzed until only incorporated counts remained inside
the dialysis bag. This material was counted directly
using Cerenkov radiation.

 

 

 

Alkaline Polynucleotide

phosphatase kinase a2P incorpor-
DNA treatment treatment ated (cpm)
gh-l DNA x - . 18
gh-l DNA - x 610
gh-l DNA x x 595
limit polymer- x - 17
limit polymer - x 1989

limit polymer x x 4421

 

118

Since each labeled DNA molecule contains only one
32? atom and the amount of DNA in each reaction is known
as well as the specific activity of the 32PeATP. the number
of nucleotides in each DNA molecule and hence molecular
weight of the DNA can be calculated. This number-average
molecular weight calculation (see appendix for actual
calculations) gave a value of 1.49 x 106 for the gh-l DNA
limit polymers and 1.2 x 107 for the whole gh-l DNA molecules.
These values are for denatured single stranded molecules
and therefore must be multiplied by 2 to compare with duplex
equivalent molecular weights. The known molecular weight for
denatured gh-l DNA is 1.25 x 107. The value for the limit poly-
mers also agrees closely with the values obtained by other
methods (see Table V).

The identity of the 5' terminally labeled nucleotides
in gh-l DNA and ghal DNA limit polymers was determined as
follows. The DNA was reduced to 5' mononucleotides by the
successive action of DNase I and venom phosphodiesterase.
To the samples in Table VIII which had been treated with
phosphatase and kinase. was added 0.1 mg of pancreatic
DNase (DNase I). They were incubated at 37° C for
30 minutes. Then. 0.1 ml 0.7 M glycine buffer. pH 9.2
was added followed by 0.4 units of venom.phosphodiesterase
(treated as described in Materials to eliminate trace
amounts of 5' AMPase activity). The reaction was carried
out at 37° C for 30 minutes after which 0.05 ml of each
of the deoxyribonucleotides (10 mgflml) was added. The

mixture was diluted to 20 ml with 320 and chromatographed

119

on a Dowex column as described in Methods.

Figure 24 shows the distribution of 32P in the nucleo-
tide peaks eluting from the column. The radioactivity
was distributed among the unlabeled nucleotide markers with
the percentages listed in Table IX. The 4% dpA and 5%
de values are high since some A and T residues were found
to be present at the termini of whole gh-l DNA molecules.
After correcting for the presence of these nucleotides the
composition at the 5' ends of gh-l DNA limit polymers
produced by the action of wheat seedling nuclease is 48%

de and 52% dpG.

Effect of Temperature on ADNA Hydrolysis

A large increase in the rate of hydrolysis of nDNA
with moderate increases in temperature had been observed
in earlier studies. In order to quantitate this effect,
the extent of hydrolysis of ADNA by wheat seedling nuclease
at 200 C and 300 C was investigated. Reactions contain-
ing 0.05 ml ADNA (30 ,ugms), 0.01 ml 0.5 M.NaAc, pH 5.0,
0.035 ml H20 and l DNase unit of wheat seedling nuclease
Type II were incubated at either 200 or 30° C for one hour.
Reactions were terminated by adding 0.015 m1 1 N NaOH and
0.03 ml 4.5 NaCl. The samples were then sedimented on
alkaline sucrose gradients as described in Methods. The
average duplex equivalent molecular weight of the products
from the 200 C incubation was 15 x 10°. The 30° C reaction

produced products with an average duplex equivalent mole-

120

 

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122

Table IX

5' terminal nucleotide composition of
gh-l DNA limit polymers

Limit polymers which had been labeled with 32P at
the terminal nucleotide position were completely degraded
to mononucleotides by the successive action of DNase I
and venom phOSphodiesterase and were then separated by
dowex chromatography as described in Methods.

 

 

 

Nucleotide 32F (cpm) Percent of total counts
de 2392 43
dpA 228 4
dpG 2647 48
dPT 297 5
de + dpG 5039 91

dpA + de 522 9

 

123
cular weight of 2 x 106. Since ADNA is 30 x 106 daltons,
the number of bonds broken during the 20° C and 30° C
incubations is l and 15 reSpectively. Since a 15 fold
increase in activity is much larger than that expected
from the thermal activation of the enzymatic catalysis

(010 effect), the increase is attributed to changes in

substrate structure.

DISCUSSION

The purification procedures reported for wheat seed-
ling nuclease were developed in order to achieve a highly
purified form of the enzyme requiring a minimum number of
days to prepare. Chromatography on a carboxymethyl cell-
ulose (CMC) column was found to be a very useful step in
the latter stages of purification. Since wheat seedling
nuclease has a much lower binding affinity to this material
at pH 5.0 compared to the other proteins present, most of
the contaminating protein was removed during this step.
When the CMC column step is used following the heat step
(Type II procedure), a large increase in the specific act-
ivity of wheat seedling nuclease results. The enzyme result-
ing from this procedure represents an increase in specific
activity of over 4.000 compared to the crude homogenate and
is better than 80% pure as judged by the protein bands on
SDS gels. This enzyme preparation , which requires three
days and was devoid of any phosphatase or phosphodiesterase
contaminating activities, provided a convenient source of
enzyme for the studies on the "mechanism of action" of
wheat seedling nuclease.

The Type III purification procedure also utilized
the CMC column step (following the phosphocellulose step)

and yielded wheat seedling nuclease in a highly purified
124

125
form which could be used for the investigation of protein
properties.

Various methods based on the determination of the
size or the size and shape of protein molecules seemed to
indicate that the Type III enzyme preparation contained
predominantly a single protein species. These methods
yielded the following results; 1) a single symmetrical
boundary observed during the sedimentation velocity ex—
periment in the analytical ultracentrifuge, 2) a single,
coincident peak of protein and enzyme activity found after
sedimenting wheat seedling nuclease Type III through a
sucrose gradient and 3) a single peak of protein and wheat
seedling nuclease activity eluted from a Sephadex G-100
column. The fact that the Specific activity remained con-
stant throughout this peak indicates a close correlation
between total protein and enzyme activity. This protein-
activity peak eluted between one and two times the column
void volume where maximum separation of protein occurs.

In addition, electrophoresis of the wheat seedling nuclease
in polyacrylamide gels in the presence of SDS gave one
major band comprising 90-95% of the total protein stained
on the gel.

The molecular weights determined from the sedimenta-
tion velocity experiments in the analytical ultracentri-
fuge and the sucrose gradient sedimentation analysis (43,000
and 45,000 respectively) are estimations based on assump-

tions regarding the shape of the protein molecule.

126
The molecular weight of wheat seedling nuclease

determined by SDS gel electrophoresis.was 43.500.

The standard proteins used to calibrate the gel system
had molecular weights from ll.000-43.000. Thus. the
estimation of size for wheat seedling nuclease requires
a slight extrapolation of the standard curve. Weber
and Osborn (44) have studied the migration of 40 proteins
in SDS gels and found that all the proteins studied fell
on the standard curve. regardless of the shape of the
protein in the native state. The conditions used dur-
ing the SDS electrophoresis method are expected to
produce single polypeptide chains. Therefore. since
this method yields a molecular weight which corres-
ponds to that determined by other methods for the

intact protein molecule. wheat seedling nuclease probably
contains one polypeptide chain.

After these indications that the preparation

was rather homogeneous. the existence of multiple

ionic forms of purified wheat seedling nuclease was

a somewhat surprising discovery. Isozymes bearing
different net protein charges have been observed

for a number of enzymes (53). Use of the term
'isozymes' to describe the multiple ionic forms of wheat
seedling nuclease is not appropriate. however. since.
only one of the 6-12 differently charged proteins.
possesses any enzymatic activity.

12?

Multiple protein species produced by protec-
lytic activities during purification of several
enzymes have been noted (54.55). In these cases. all
of the protein species possessed enzymatic activity.
However. when the esterase inhibitor. phenylmethyl-
sulfonyl fluoride (PMSF) was added during the puri-
fication. the multiple forms of the enzyme were elimi-
nated yielding one active protein. When wheat seed-
ling nuclease was prepared in the presence of PMSF.
the same pattern of'multiple protein bands remained
after polyacrylamide gel electrophoresis of the
purified enzyme. Furthermore. the specific activity
of the purified enzyme was essentially the same with
or without PMSF present during purification. The
possibility remains. however. that wheat seedling
nuclease is modified by a proteolytic enzyme(s) which
is not inhibited by the general protease inhibitor
PMSF. The fact that the inactive components of the
wheat seedling nuclease preparation appear to be at
least as large. if not larger. than the active pro-
tein. would indicate that the enzyme has not under-
gone extensive proteolytic digestion.

Another consideration which might explain the pre-
sence of the multiple. inactive forms of wheat seedling
nuclease is chemical alteration of the enzyme during puri-

fication. This possibility does not seem too likely. since

128
no greater than 50% of the total DNase activity is lost
during any individual step in the purification procedure,
yet the active protein band on polyacrylamide gels com-
prises less than 10% of the total protein present in all
of the bands.

The electrophoresis experiments with purified wheat
seedling nuclease on gels containing various percentages
of acrylamide indicate that all of the protein species
are approximately the same size. A slight increase in
molecular weight (about 2,000) was observed for the more
negatively charged proteins, although this value is cer-
tainly not much larger than the experimental accuracy
for this procedure. The active band represents one of
the least negatively charged proteins with only one pro-
tein band(comprising just a few percent of the total
protein on the gel) being more positively charged. Neg-
atively charged, non-protein moities associated with
wheat seedling nuclease could account for these obser-
vations assuming that binding to these moieties caused
complete inactivation of the enzyme.

The low AZBO/Azao ratio for wheat seedling nuclease
may be due to a non—protein group associated with the
enzyme. Analysis of the aromatic amino acid groups pre—
sent in wheat seedling nuclease indicates that the low
Azeo/Azgo ratio is probably not due to an abnormal dis—
tribution of aromatic amino acids. Furthermore, the fluo-

rescence data indicate the possible presence of a

129
chromophore in wheat seedling nuclease which absorbs light
energy at 255 nm but which does not fluoresce.

The possible association of nucleic acid components
with the wheat seedling nuclease is consistent with the
spectral data. Nucleic acids absorb light energy in the
ultraviolet region of the spectrum but do not produce any
fluorescence at neutral pH (56). Assuming that nucleotides
and/or oligonucleotides are bound to wheat seedling nuclease,
the observed Aaao/Aggo ratios of 1.09 to 1.30 would cor-
respond to two to four moles of nucleotide per mole of
enzyme based on average extinctions for proteins and nu-
cleic acids at 280 nm and 260 nm.

The isoelectric focusing experiment on purified
wheat seedling nuclease resulted in the focusing of only
a small amount of protein, along with all of the activity,
at pH 5.2. The remainder of the protein focused at pH
2.0. This pH is well below that of any ionizable group
in any of the common amino acids. The phosphate ester
group in nucleotides and/or oligonucleotides, however,
has an ionizable hydrogen with a pK near this value.

Other evidence consistent with the presence of a
negatively charged ligand which absorbs light energy at
260 nm is seen from the A280 and A250 scans of multiple
protein bands on polyacrylamide gels. As the four major
protein bands become more negatively charged, their
Agog/A260 ratio decreases. In addition, it can be seen

from the plot of relative mobility versus percent

130
acrylamide in the gel that the various protein bands
appear to be separated by roughly equal charge increments
(vertical displacement on the ordinate scale). It is
interesting, then, to speculate that the various ionic
forms of wheat seedling nuclease arise from the binding
of integral multiples of negatively charged ligands.

The following model system is offered as one expla-
nation for the existence of multiple, inactive forms of
wheat seedling nuclease. Nucleotides and/or oligonucleo-
tides might be bound to the enzyme yielding a variety
of inactive proteins containing different net charges.

If this occurs in the cell, it might represent a control
mechanism by which the cell is able to limit nuclease
activity in certain situations.

If negatively charged ligands are indeed bound to
wheat seedling nuclease, they are either very tightly
bound or are covalently linked, since extensive dialysis
produces no change in the band pattern on polyacrylamide
gels. Polyguanylic acid is not hydrolyzed by wheat seed-
ling nuclease but, never the less, binds tightly to the
enzyme as demonstrated by inhibition studies. Furthermore,
it has a Amax at 255 nm which is the same wavelength as
the peak produced by subtracting the enzyme's absorbance
(which produces fluorescence) from the absorption spect—
rum of wheat seedling nuclease. It might therefore be
possible that oligomers of guanylic acid are bound tightly

by the enzyme - perhaps buried in the protein's interior

131
by a conformational change in the protein's structure
3 during binding. Another explanation for the presence
of a non-dialyzable ligand would be a covalent enzyme-
1igand bond produced by another enzyme activity in the
cell. Glutamine synthetase is an example of an enzyme
containing adenylic acid covalently linked by a separate
enzyme activity (57).

As pointed out earlier in this thesis, the mechanistic
studies conducted on wheat seedling nuclease were limited
to those areas which established an unusual or unique facet
of enzymatic catalysis compared to mechanisms of other
nucleases reported in the literature. For example, wheat
seedling nuclease appeared to have the ability to hydrolyze
dDNA by an endonucleolytic mode of cleavage while degrad-
ing rRNA exonucleolytically.

The conclusion that the DNase activity is endonucleo-
lytic is based on the observations that gel filtration of
early reaction products showed the presence of large oligo-
nucleotides, while extensive hydrolysis yielded a mixture
of small oligonucleotides and mononucleotides (16). However,
these observations do not rule out the possibility of an
additional exonucleolytic component to the DNase activity.

Gel filtration of early stages of rRNA digests reveal-
ed primarily the presence of small products (presumably
mononucleotides) although some large products were also
observed. Since rRNA has a very complicated secondary
structure. these results are difficult to interpret. For
example. perhaps small products were being produced from

132
small single-stranded. looped areas in the rRNA with areas
of secondary structure remaining intact. The experiment
in which polyadenyl ic acid was hydrolyzed by wheat seed-
ling nuclease and assayed concurrently for 5'-AMP and acid-
soluble material. provided a more quantitative evaluation
for the mode of cleavage on a better defined RNA substrate.
These results indicate that primarily mononucleotides are
released during hydrolysis of poly A. although the presence
of a small amount of endonucleolytic activity cannot be
ruled out. These results tend to indicate that DNA is cleaved
primarily endonucleolytically by wheat seedling nuclease.
while RNA is. for the most part degraded by an exonucleo-
lytic mode of cleavage.

The monophosphatase activity associated with wheat
seedling nuclease has been referred to as a 3'-nucleotidase
activity. This is possibly a slight error in nomenclature
since 2'-UMP is also dephosphorylated to a small extent.
However. none of the other 2'-ribonucleoside monophosphates.
nor any of the 5' isomers of the ribo- or deoxyribonucleo-
side monophosphates are hydrolyzed by wheat seedling nuclease.
The small amount of activity associated with 2'-UMP could be
due to the presence of 3'-UMP in the commercial preparation.
Therefore. it is still felt that '3'-nucleotidase' is the
most descriptive term.

The pH profiles for the 3'-nucleotidase activity
are considerably different for the four 3'-ribonucleoside

monophosphate substrates (A). OD. UP. GP).

133
This probably reflects the different extent

of ionization of the various charged groups in the hetero-
cyclic bases of the nucleotides at the various pH values.
The pH profiles are rather broad for 3"AMP, 3'-CMP and
3'-UMP, whereas 3'—GMP hydrolysis shows a pH optimum

at pH 7.9. Thus at pH 5-6.4 the relative order of hydro-
lysis is 3'-AMP>3'-UMP7 3'-CMP>3'-GMP. On the other
hand, at pH 7.3-8.5, the relative order is 3'-AMPt73'-GMP7
3'“UMP>'3'-CMP. This observation probably explains the
discrepancy observed in the relative order of hydrolysis
of the 3'-ribonucleoside monophosphates by a similar
enzyme, mung bean nuclease. For this enzyme, Welters

and Loring (26) published the following order which was
determined at pH 8: 3'-AMP'>3'-GMP73'-UMP>3'..CMP.
Laskowski (28), at pH 5, observed the same order as that
for wheat seedling nuclease at the same pH - namely,
3"AMP v 3'-UMP v 3'-CMP‘I 3'-GMP.

Because of their complex substrates, kinetic anal-
ysis of nucleases has largely been avoided. When nucleic
acid polymers are degraded during the course of nucleases
catalyzed hydrolysis,the substrates continually change
and inhibitor products may form, producing autoaccelera-
tion or autoretardation (58). Furthermore, in most cases
an initial collection of homogeneous polymeric molecules
is impossible to obtain. For these reasons, the kinetic
analysis of initial rates is difficult to interpret.

Wheat seedling nuclease possesses a 3'-nucleotidase

134
activity which is amenable to study by classical kinetic
methods. The assay methods available for this type of
activity, however, are fixed-time assays which are diffi-
cult and tedious methods for obtaining kinetic data.
Therefore, a continuous spectrophotometric assay was de-
veloped which could be used to measure initial rates of
3"AMP dephOSphorylation. This assay uses adenosine de-
aminase to convert the adenosine produced from 3'eAMP
by the action of wheat seedling nuclease to inosine, which
has a much lower molar absorptivity at 265 nm. This assay
technique could have general utility for a number of
nucleotidase activities which dephosphorylate any of the
isomers of adenylic acid.

The Km and Vmax values obtained for the hydrolysis
of 3'vAMP indicate that it could be a natural physiolog-
ical substrate for wheat seedling nuclease. The mono-
nucleotide, 5'-AMP, which is not hydrolyzed by wheat seed-
ling nuclease, was seen to act as a competitive inhibitor
towards the 3'-AMPase activity. ‘When the phosphodiester-
containing non-substrates, 2',3'-cyclic AMP and ApA were
included in the reaction mixture, non-competitive inhi-
bition of the 3'sAMPase activity was observed. This is
an interesting result in light of the fact that wheat
seedling nuclease has the ability to hydrolyze two types
of substrates - namely, the phosphodiester molecules,

RNA and DNA and the phosphomonoestar-containing 3'-nucleo-

side monophosphates.

135

The inhibition of the 3'-AMPase activity by NaCl
appears to be a general salt effect since other chloride
salts, as well as inorganic phosphate, are also inhibitory.
The inhibition probably represents the binding of the salt
ions to charged groups on the enzyme at or near the active
site. This type of inhibition has been observed for sev-
eral other enzymes (58,59). Since all the chloride salts
were approximately equally effective inhibitors, the inhi-
bition may be explained by the binding of Cl- ions to a
positively charged center(s) which also binds the sub-
strate (s). It is again interesting that two different
types of inhibition were observed: one for the 3'-nuc1eo-
tidase activity and the other for the RNase activity.
NaCl inhibits the hydrolysis of 3'-AMP competitively while
producing a noncompetitive inhibition for the pApA
hydrolysis.

While kinetic studies .are seldom able to establish
a particular mechanism unequivocally, they can often aid
in suggesting mechanistic possibilities. ‘Wheat seedling
nuclease contains three distinct catalytic activities:
DNase; RNase; and 3'-nucleotidase activities. The kinetic
data suggest some interesting possibilities for the inter-
relationship of these activities. One possibility con-
sistent with the data, is that the 3'-AMPase (3'-nuc1eo-
tidase) activity and the pApA hydrolysis (RNase activity)
occupy separate and distinct active sites on the wheat

seedling'nuclease molecule. This would explain the

136
noncompetitive and competitive inhibition by phosPhodiester
and phosphomonoester inhibitors,respectively, towards
the 3'-AMPase activity. This 2-site model is also con-
sistant with the fact that NaCl inhibits the 3'vAMPase
activity competitively while acting as a noncompetitive
inhibitor towards the hydrolysis of pApA. Another pos-
sibility is that the phosphodiester-containing inhibitors,
2',3'-cyclic AMP and ApA, are not binding to the pApA
site, but are binding to another site - possibly the
DNase site or to a separate allosteric or effector site.
Finally, the possibility exists that the three activities
are all contained in subsites of the same active center.
In this case, for example, the C1- ion inhibition could
be explained if Cl- competed for the positively charged
center(s) which binds 3hAMP, but bound very tightly to the
center(s) or configuration responsible for binding pApA.
If this were the case, pApA may not be able to "compete
out" the Cl_ ion, until very high concentrations of pApA
are obtained, and thus demonstrate a type of non-competi-
tive inhibition. To test this type of noncompetitive
inhibition, substrate concentrations well above the Km
need to be tested. unfortunately, this data could not
be obtained since the pApA reaction has a high Km and the
pApA concentration in the assay is limited to that amount
which produces an A275 below 2. This fact also explains
why the ultraviolet absorbing inhibitors could not be

tested in the pApA hydrolysis reaction.

137

The fact that the relative order of base preference
expressed by the enzyme is the same for all three activities
is, perhaps, not surprising if one makes the assumption
that all three activities are catalyzed by the same general
active center. The data from experiments on the production
of the four different mononucleotides during the hydrolysis
of dDNA, as well as the hydrolysis of the four different
ribohomopolymers indicate a reluctance of wheat seedling
nuclease to hydrolyze phoSphodiester bonds adjacent to
guanylic and cytidylic acid residues. The ribohomopolymer
data should be interpreted with some caution, however,
since the polymers exhibit different degrees of ordered
structure with the given conditions of the assay. At a
value near the pH optimum for the DNase and RNase activities
(pH 5), the relative order of base preference for the 3'-nucleo-
tidase activity is the same as that for the RNase and DNase
activities. In fact, when the 3'-deoxyribonucleoside mono-
phosphates are used as substrates, 3'-dGMP and 3'-dCMP are
not hydrolyzed at all. As was pointed out, however, the
base preference for the 3'-nucleotidase activity changes
at higher pH - at least in the case of the 3'-ribonuc1eo-
side monophosphates.

Perhaps the most exciting area of investigation was
the hydrolysis of native DNA molecules by wheat seedling
nuclease. Early work with wheat seedling nuclease demon-
strated a large difference in the rate of acid-soluble

products produced when native and denatured DNA were

138

hydrolyzed by the enzyme. When the limited hydrolysis
of native DNA was examined by measuring the size of the
products produced during the course of the reaction, it
was found that wheat seedling nuclease catalyzed the hydro-
lysis of only a limited number of phosphodiester bonds
in large DNA molecules, producing "limit polymers" still
of high molecular weight, which were resistant to further
hydrolysis by additional enzyme over extended periods
of time. This somewhat surprising result indicated that
there may be a few, highly specific sites or areas in
the native DNA molecules which are recognized and cleaved
by wheat seedling nuclease.

A number of methods were used to investigate the
extent and nature of this apparent site specificity.
High molecular weight g, 22;; DNA or intact, linear-duplex
viral DNA of known molecular weight was used for these
studies. Since the average molecular weight of the limit
polymers produced by wheat seedling nuclease is also known,
the number of double-strand scisflons made in the original
DNA can be estimated. When this estimation is made for
all of the DNA's tested, the result is one double-strand
cleavage for every 6,100 to 9,090 phosphodiester bonds
present in the original DNA, or one double-strand scission
per 3,500-4,545 nucleotide pairs. In other words, for
example, gh-l DNA (MW==23 x 10°) is cleaved about seven
times to yield limit polymers of 3 x 10° daltons.

An analysis of the acid soluble products produced

139
during the limit digestion of 32p gh—l DNA, revealed that

the acid-soluble material was near the borderline of de-
tectability. The sensitivity of the method was such that
0.1% conversion to acid-soluble products would be just
detectable. Since this value would correspond to only
seven nucleotides per cleavage, little, if any, acid-sol-
uble material is released during the limit digestion of
native gh-l DNA. This fact obviously contradicts the early
results obtained with commercial calf thymus and salmon
sperm DNA. A possible explanation for the small amount
of acid-soluble production observed with these substrates
is the contamination with single-stranded DNA, RNA, or
loose, single-stranded ends produced from physical
shearing of the DNA in preparation, shipment and storage.
Also, the contamination of the commercial DNA with other
nucleolytic enzymes could explain the appearance of acid-
soluble products.

Wheat seedling nuclease was judged to produce only
double-stranded cleavages in native DNA for the following
reason. After gh-l DNA or A DNA was digested to limit
polymers, it yielded the same duplex-equivalent molecular
weight in both neutral and alkaline sucrose gradients.
The alkaline sucrose gradient technique sediments DNA
as single,randomly-coiled strands, whereas the neutral
sucrose gradient technique sediments the DNA as hydrogen-
bonded double-strands. Therefore, the equivalence in

molecular weight indicates the absence of any "nickase"

140

activity or single-strand interruption in the limit polymers.

The data collected by labeling the 5' termini of the
gh-l DNA limit polymers can be used to evaluate several
parameters of the wheat seedling nuclease activity. First
of all, since only one nucleotide is labeled per single-
stranded DNA polymer, it is possible to calculate the average
number of nucleotides in the limit polymers and hence the
number-average molecular weight. Furthermore, this data also
indicates the number of new termini produced during the
limit digestion and therefore the number of cleavages cata-
lyzed by the enzyme. Finally, by separating the labeled
nucleotides, it is possible to identify the nucleotide present
on the 5' ends of the limit polymers which were produced
by the hydrolysis of wheat seedling nuclease.

Treatment of the data in this manner for the experi-
ments with the limit digestion products of gh-l DNA gave
the following results (see the appendix for the calcula-
tions). An average of 6.7 cleavages were produced in gh-I
DNA (MW 23 x 10°) yielding limit polymers containing 8.54
x 103 nucleotides which corresponds to a number-average
molecular weight of 2.98 x 10°. The nucleotide composition
at the 5' ends of the limit polymers was 52% G and 48% C
after subtracting the contribution of the nucleotides pre-
sent initially at the 5' end of gh-l DNA. The possible

significance of these results is discussed later.

141

There existed the possibility that wheat seedling
nuclease could recognize a large area of DNA, perhaps
several hundred nucleotides in length, which was struc-
turally unique because of an abndrmal nucleotide sequence.
In order to test this possibility, venom diesterase was
allowed to cleave an average of 118 nucleotides from the
3' end of 32P-1abeled gh-l DNA limit polymers. An anal-
ysis of these nucleotides revealed that there was an
enrichment of several mole-percent in the A-T content,
compared with that of the whole gh-l DNA molecule. This
enrichment in A-T does not seem sufficiently great
to cause a change in the DNA structural conformation.
However, the possibility remains that this value reflects
a much higher enrichment in A-T in a much smaller area
near the cleavage site.

Another interesting aspect of the hydrolysis
_of native DNA by wheat seedling nuclease is the separation
of the specific cleavage sites along the DNA molecule.
The limit pelymers produced by treatment of gh-l DNA
with wheat seedling nuclease appear to be roughly the
same size, indicating a regular or periodic spacing of
the cleavage sites in the DNA. The homogeneity of the
size of the gh-l DNA limit polymers is suggested by the
similar molecular weight values obtained by sucrose gra-
dient analysis (weight-average molecular weight) and by
the 5' end labeling experiment (number-average molecular

weight). Furthermore, the agarose gel electrophoresis

142
of the limit polymers reveals that about 90% of the poly-
mers are within roughly 10.5 x 10° daltons of the ¢-x174
marker DNA (MW=3 x 10°).

The fact that the rate of hydrolysis of g,‘ggli_DNA
is first order throughout the reaction suggests that all
of the cleavage sites are hydrolyzed at approximately
the same rate. This result would be expected if all of
the cleavage sites were identical in nature.

The supposition that an A-T rich area may be involved
at the cleavage site in native DNA is suggested by several
lines of experimental evidence. Although native DNA is
hydrolyzed at a much slower rate by wheat seedling nuclease
than is single-stranded or denatured DNA, the synthetic
duplexes, poly d(A-T) and poly rA,U, are hydrolyzed at rates
comparable to denatured DNA. On the other hand, the duplex
poly dG-poly dC is not hydrolyzed by wheat seedling nuclease.
The rapid hydrolysis of poly d(A-T) may reflect the rela-
tively weak bonding of the A-T base pairs which holds the
two self-complementary strands together (60).

Poly d(A-T) existed primarily in the duplex form in
all the assay conditions, since they were performed well
below the Tm in all cases. As the conditions were changed to
those favoring denaturation (low ionic strength and low
pH), a large increase in the rate of hydrolysis was noted,

although the synthetic polymer was still mainly in the

143

duplex form. These observations probably indicate that
for poly d(A-T), at least, the enzyme acts on single-strand
areas produced'by disruption of the secondary structure.

Other experimental data which seems to implicate
the involvement of A-T rich areas at the cleavage site
in native DNA is the early and rapid production of A
halves during the hydrolysis of A DNA. The A-T rich region
near the center of A DNA has been well characterized (61).
Inman and Schnos (61) have demonstrated with electron
microscopic pictures that when A DNA is partially denatured
at high pH, the A-T rich region in the center tends to
loop out, appearing as two single-stranded chains for a
short distance. Wheat seedling nuclease may be recogniz-
ing this area as being single-stranded in nature.

As previously mentioned the 3' end analysis revealed
only a slight increase in the mole-percent of A4'T over
an area representing an average of 118 nucleotides from
the limit polymer terminus. If all of this enrichment
were due to an area of only 6-12 nucleotides, say, from
the end of the limit polymer, then this area would be enriched
20-40% in Ai-T, compared to the whole gh-l DNA molecule.
Thus, while there does not appear to be a significantly
large abberration in nucleotide content in the general
area of the cleavage site, the possibility of a high

A-T area in the immediate vicinity of the cleavage

144
site cannot be ruled out.

As pointed out earlier, the fact that the rate of
hydrolysis for ADNA is increased by 15 fold between 200
and 300 C, suggests a structural effect of the increased
temperature on the substrate. Such an effect might be
expected if an A-T rich region were involved at the clea-
vage site. Such an area would be eXpected to undergo
partial denaturation, thus shifting the dynamic equili-
brium (single-strand configuration ridouble-strand config-
uration) to the left, thereby providing a much better
substrate for wheat seedling nuclease. This would be anal-
ogous to the large increase in rate noted for poly d(A-T)
as the ionic strength and pH were lowered.

The conceptual idea of "structural breathing" has
been noted by von Hippel and Felsenfeld (62). These authors
postulated that at temperatures below the Tm, regions
rich in A-T undergo strand separation to a greater
extent than areas rich in G-C. Therefore, it is possible
that the single-strand specificity of wheat seedling nuclease
endows the enzyme with the ability to recognize A-T rich
areas in native DNA.

It is interesting to compare the base preference
exhibited by wheat seedling nuclease
with the possibility of the involvement of A-T rich areas
near the cleavage site. The hydrolysis of the phosphate
bond adjacent to A and T was seen to be preferred to a

considerable extent compared to bonds adjacent to C and G

145
residues in dDNA, ribohomopolymers and 3'-nucleoside mono-
phosphates. Thus it may be that this base preference
is acting in tandem with the enzyme's ability to recognize
structural difference in the nucleotide composition of a
given region of DNA in order to produce a specific site
on the native DNA molecule which is recognized by wheat
seedling nuclease.

It is once again tempting to construct a model, for
the sake of discussion, which attempts to coalesce the
variety of experimental information into a hypothetical
explanation which is consistent with a maximum number of
the observed experimental results. The following model,
then, is offered for the production of limit polymers from
native DNA by wheat seedling nuclease.

In the model illustrated below, there is a small
A-T area which is diagramatically drawn to illustrate the
"structural breathing" phenomenon. Wheat seedling nuclease
catalyzes the hydrolysis of one of the A-T bonds (arrows
with asterisk) on both the upper(U) and lower (L) strand
where the single-strand character of the DNA is the greatest.
The loose A-T ends are then hydrolyzed (arrows) producing
5' phosphoryl terminated products, until a G-C pair is
encountered. At this point the enzyme is unable to bind
to the double stranded DNA molecule and is released by the

substrate.

146

1’

l
fiiplkn‘fi ,/ .
5" ' ' poXpGp 4pGpoX' ' ' ' 3 U

3'----x.xpc° .c.x.x.---5'L
f"4 9‘
7 twin4 '\

T ’\

‘fl

This model predicts that the 5' termini of the limit
polymers should contain 50% G and 50% C residues which
agrees with the experimental evidence. It also predicts
that the 3' terminus on both the L and U strands should
be either A or T. This would be consistant with the ob-
servation that the bonds adjacent to G and C residues are
resistant to attack by wheat seedling nuclease. This model
also predicts that a small amount of acid-soluble material
should be released in the form of 5'-AMP and 5'-TMP.

The A-T area drawn in the model would only release 8 mono-
nucleotides in the limit polymer. This value was about
the limit of detectability in the 32P-gh-l acid-soluble
experiment. The experimental values of 0.04% and 0.07%
acid-soluble products could easily be in error by as much
as 1 0. 04%.

Other possibilities exist, of course, to explain the
specificity indicated for the cleavage of native DNA by

wheat seedling nuclease. If no acid-soluble material

147
is released during hydrolysis, the cleavage configuration

would have to be of the following sort:

i
5 ' ---poXpC/GpC/GpoX---3 '
3 ' ----XpoC/Gs}C/GpoXp--5 '

where C/G indicates the presence of either C or G. There-
fore, this would necessitate the cleavage of a CpG or GpC
bond in order to explain the 50% G and 50% C 5' terminal
data if the double-strand scission was made directly across
the duplex chain (i.e. if the cleavage was not staggered).
This possibility seems less likely since the base prefer-
ence data suggest a considerable reluctance by wheat seed-
ling nuclease to hydrolyze bonds adjacent to C and G
residues.

Other areas in the DNA molecule besides A-T rich
regions may play a role in the site recognition. For
example, symmetry elements in the DNA molecule or unique
base sequences may be responsible for the recognition of
certain areas by wheat seedling nuclease. In addition, the
possibility remains that the binding of wheat seedling nu-
clease to native DNA may contribute to the opening of the
duplex to permit hydrolysis of the single stranded areas.

The data pertaining to the transcription by the
phage-induced, DNA-dependent RNA polymerase from the limit
polymer templates is interesting for several reasons.

First, the fact that the Km and Vmax values are similar

148

to those obtained when whole gh-l DNA is used for a temp-
late, demonstrates a high catalytic efficiency for the
limit polymer templates. This would seem to indicate
that either the promotor and terminator regions were pretty
much intact (at least functionally) in the limit polymers
or that they are not required to affect efficient trans-
cription. Also it is known7 that the phage induced poly-
merase transcribes guanylic acid as the first residue in-
corporated into RNA chains. This fact may be significant
since the limit polymers end in 50% C and 50% G.

It is particularly intriguing to speculate about
the significance of the manner in which wheat seedling
nuclease degrades native DNA molecules. Although the
mechanism of native DNA hydrolysis may be purely happen-
stance,and therefore of no significance to the cell, this
seems unlikely due to the unusual specificity involved.
Since wheat seedling nuclease produces only a limited num-
ber of double-strand cleavages which are spaced along the
DNA molecule at average intervals, comparable to individual
cistrons in size, a mechanism for site-specific recombi-
nation could be envisaged. Such a mechanism has been demon-
strated‘ip‘yigyp for the restriction endonuclease, R I
(63). In this case, the specific cleavage produced by the
endonuclease results in a number of fragments which can be
recombined by a separate ligase activity to produce a new
population of recombinant molecules. H. Sobell has also

proposed a molecular mechanism for genetic recombination

 

7Personal communication from Dr. J. Boezi.

149
which postulates a specific double-strand cleavage by an
endonuclease (64). In these respects, it is interesting
to note that, in the case of gh-l DNA, wheat seedling
nuclease produces fragments which correspond in size to the
average transcriptional unit of the phage DNA and which
retain a good portion of their template activity.

Although no modification activity has been demon-
strated to be associated with wheat seedling nuclease,
restriction of foreign DNA molecules might also be a func-
tion of the action of wheat seedling nuclease on native
DNA. As noted in the introduction to this thesis, the
bacterial restriction endonucleases also demonstrate the
same high degree of specificity towards the hydrolysis of
native DNA.

The fact that wheat seedling nuclease appears to
possess the ability to recognize certain structural and/or
chemical sites in a double-stranded DNA molecule, suggests
a role for the enzyme as a probe of DNA structure. R. D.
Wells (65) has studied the physical and chemical properties
of a number of synthetic DNA molecules of known base
sequence. He goes on to postulate that areas of abnormal
base sequence in naturally-occurring DNA's may be involved
in structure-function relationships. For example, areas
rich in A-T have been demonstrated to be involved at the
insertion site for A prophage in g, 221; DNA (66) and at
the sites in T5 and T7 where g, ggii'RNA polymerase binds

(promotor sites) (67). The operator region in A DNA, to

150
which the lactose repressor binds,has recently been demon-
strated to have a unique base sequence (68). Therefore,
enzymes such as wheat seedling nuclease, which are able
to recognize certain specific areas in DNA may have poten-

tial use as probes of these structure-function relationships.

 

SUMMARY

Wheat seedling nuclease has been purified over
10.000 fold with a 5% yield of activity. This procedure pro-
duced 0.5 mg of protein from 7 kilograms of wheat. The
isolated preparation was Judged to be nearly homogeneous
in regards to size and shape as indicated by the following
results: a) sedimentation in the analytical ultracentri-
fuge revealed a single. symmetrical boundary. b) sedimenta-
tion through a sucrose gradient resulted in a single.
coincident peak of protein and enzyme activity. c) filtra-
tion in Sephadex 0-100 yielded a single protein-activity
peak. throughout which the activity remained constant. and
d) polyacrylamide gel electrophoresis in the presence of
2-mercaptoethanol and SDS revealed the presence of only one
major protein band comprising greater than 90-95%'of the
total protein stained on the gel.

However. when electrophoresis of this preparation
of wheat seedling nuclease was performed at pH 8 in the
absence of SDS and z-mercaptoethanol. 6-12 protein bands
were detected. Furthermore. the 3 activities associated
with wheat seedling nuclease were associated with only one
of these bands. The band responsible for the enzyme act-
ivities was one of the least negatively charged bands and

151

152
contained less than 10% of the total protein stained on
the gel. All of the multiple-ionic forms of wheat seedling
nuclease were determined to be approximately equal in size.
This conclusion is based on the fact that when the percent
acrylamide contained in the gel was plotted as a log-
arithmic function of the relative mobility, similar slopes
were obtained for all of the bands.

The molecular weight of wheat seedling nuclease
was determined to be approximately 43,000 as determined
by the following methods: a) sedimentation velocity exper-
iments on the analytical ultracentrifuge, 9) sucrose gra-
dient sedimentation with proteins of known molecular weights,
c) SDS gel electrophoresis with a system calibrated with
standard proteins. Since the SDS electrophoresis procedure
yielded a molecular weight which was similar to the other
two methods, it is likely that wheat seedling nuclease
exists as a single polypeptide chain.

Wheat seedling nuclease has an Agao/Aggo ratio of
1.1-1.3 which is lower than that observed for most proteins,
This ratio may represent the presence of a non-protein
group bound to the enzyme since the aromatic amino acid
content cannot explain this low Agao/Ageo ratio.

Wheat seedling nuclease has been demonstrated to
possess the ability to degrade denatured DNA by an endo-
nucleolytic mode of cleavage yielding 5'-phosphoryl term-
inated mono— and oligonucleotides (16). When polyriboade-

nylic acid is degraded by wheat seedling nuclease, nearly

153
all of the products released throughout the reaction are
5'-AHP. indicative of an exonucleolytic mode of hydrolysis.

Kinetic analysis of the rates of hydrolysis of 3'-AMP
and pApA by wheat seedling nuclease was perfonmed by using
a continuous. spectrophotometric assay developed for this
purpose. The assay was based on the decrease in absorbance
produced when either adenosine or 5'-AMP (products of the
reaction catalyzed by wheat seedling nuclease) was deaminated
by a specific amino hydrolase coupling enzyme. The Km and
vmax values obtained for the dephosphorylation of 3'-AMP
were 1.5 x 10‘5 M and 1.3 x 102 jimoles/min/mg respectively.
The hydrolysis of pApA yielded Km and vmax values of
9.1 x 10'5 M and 1.67 x 103 ;imcles/min/mg.

Kinetic inhibition studies were conducted using
3'-AMP as the substrate. 5'-AMP. a molecule containing
a single phosphomonoester linkage. was found to be a com-
petitive inhibitor while the phosphodiester compounds.
2'.3'-cyclic AMP and ApA. were found to inhibit the reaction
in a noncompetitive manner.

A general salt inhibition was noted for wheat seedling
nuclease. The 3'-AMPase reaction was inhibited competitively
by NaCl. However. when NaCl was included in the pApA
hydrolysis reactions. noncompetitive inhibition resulted.

A consistent order of base preference for all three
of the activities associated with wheat seedling nuclease
was observed at pH 5.0. This preference was found to be
A >T(U) > 0 >0. Phosphodiester bonds adjacent to cytidylic

and guanylic acid residues were found to be quite resistant

15h
to cleavage by wheat seedling nuclease.

When high molecular weight. native g. 92;; DNA or
intact. linear-duplex viral DNAumolecules are hydrolyzed
by wheat seedling nuclease. only a few. specific double-
stranded cleavages occur. The resulting ”limit polymers.“
-still of high molecular weight - are resistant to further
hydrolysis by wheat seedling nuclease. **k

The nature of the sites on the duplex DNA recognized g
by the enzyme was investigated. The production of limit ‘
polymers (MW = 3 x 106) from gh-l DNA (MW - 23 x 106) was

 

L
accompanied by the release of 0.0h-0.08$ acid-soluble mat- '
erial. This corresponds to less than 8 nucleotide equiva-
lents released as acid-soluble material for each limit
polymer produced by the action of wheat seedling nuclease.
The 5' termini of limit polymers labeled with 32F by the
action of polynucleotide kinase contained either cytidylic
or guanylic acid but no adenylic or thymidylic residues.
Wheat seedling nuclease exhibits a very large preference
for denatured DNA as compared to native. double-stranded
DNA. The fact that wheat seedling nuclease can hydrolyze
the double-stranded. synthetic polymer. poly d(ApT) at rates
comparable to the hydrolysis of denatured DNA. indicates
that wheat seedling nuclease may recognize ApT rich areas
in native DNA. This contention is further supported by
the following evidence: a)A.DNA. which contains an ApT rich
region near its center. is rapidly cleaved to produce ).halves.
b) wheat seedling nuclease possesses a considerable pre-

ference for bonds adjacent to A and T residues while showing

155
a marked resistance to the hydrolysis of bonds adjacent
to C and 0 residues. c) the nucleotide composition of an
area within 118 nucleotides of the 3' end of the gh-l DNA
limit polymers showed a slight increase in ApT content as
compared to the whole gh-l DNA molecule and d) the hydrolysis
rate of native )xDNA increased 15 fold between 20° and 30° C
indicating a major structural effect of the increased
temperature on the substrate.

The biological significance of the wheat seedling
nuclease remains to be established: however. the enzyme's
catalytic properties are compatible with functions such
as restriction. that is. the recognition and cleavage of
foreign DNA's - or genetic recombination. Finally. wheat
seedling nuclease may be of current importance as a probe

of native DNA structure.

APPENDIX

APPENDIX A

Abbreviations

A,G,U,T,C,X - nucleosides (adenosine, guanosine, uridine,
thymine, cytidine, x any of these nucleotides)

dA, dG, dX - deoxyribonucleosides

pApA - adenylyb(5',3')adenosine 5'-phosphate

2'-NMP, 3'-NMP, 5'-NMP - isomers of the nucleoside monophos-
phates

2'-dNMP, 3'-dNMP, 5'-dNMP - isomers of the deoxyribonucleo-

side monophosPhates.

poly d(A-T) - alternating copolymeric duplex of A and T

poly dG-poly dC - homopolymeric duplex of poly dG and poly dC

poly rA,U - random copolymer of equal amounts of A and U

rRNA - ribosomal RNA

dDNA - denatured DNA

nDNA - native (double-stranded) DNA

DEAE- - diethylaminoethyl-

TEAB - triethylammonium bicarbonate

SDS - sodium dodecalsulfate

BSA - bovine serum albumin

TCA - trichloracetic acid

tris-HCl - tris(hydroxymethyl) aminééthane titrated to the
given pH with HCl

NaAc, Zn(Ac)2 - sodium and zinc acetate

CMC - carboxymethyl cellulose

PC - phOSphocellulose

Pi, PPi - orthophosphate, pyroPhosphate

A,90Dx unit - amount of material dissolved in 1.0 ml to

give an absorbancy of 1.0 at the wavelength,
A, in a cuvette with a 1 cm lightpath

01° - the increase in an enzymatically catalyzed reaction
for a given 10° C increase in temperature

156

APPENDIX B
Growth Media

E. coli c.00xc1t8875 medium:

 

Per liter of 6.8 buffer:

5.0 gm casamino acids (0.5% final conc.) - autoclave
for 15 minutes, then add aseptically

Add 0.25 ml of thiamine (8;) 4 mg/ml H20 - this has
to be filter sterilized, cannot be autoclaved.

Add 20 ml of 25% glucose in H20 - sterilize this

by itself, then add it aseptically

6.8 buffer: per liter distilled H20

KZHPO4 8.7 gm
I012PO4 6.8 gm
(NI-I4)2Cl 1.0 gm
MgSO4-H20 0.1 gm
CaC12 (20 mg/ml stock) 0.25 gm
FeC13-7H20 (1 mg/ml stock) 0.25 ml

Adjust pH to 6.8 with KOH or KCl
Add CaC12 solution drop wise as the last addition
(i.e. after everything else is in solution) with
stirring to prevent precipitate of CaPO4

E.coli infected with T, phages

Per liter:

 

NH4C1 1 gm
Naanpo. 5.5# gm
KH2P04 3 gm
NaCl 5 gm
M9504 0.5 gm
Casamino acids 10 gm
glucose 4 gm
Pseudomonas putida - low phosphate medium
C medium phosphate* 600 ml
KH2P04 (0.01 M) 50 ml
tris-HCl (1.0 M, pH 7.5) 80 ml
Casamino acids 5 gm
glucose (20%) 100 m1
*C medium minus phosphate .
NH4C1 2 gm
KCl and NaCl (each) 4 gm
Mgc12 (1 M) 0.” ma
Na2804 (0.2 M) 10 m ml
H20 600 m1

FeCla, Ca(N03)2, MnSO4, NaMoo. trace amounts
157

APPENDIX C
Calculations
Tyrosine and tryptophan content in wheat seedling nuclease
The two unknowns, Ntrp (the number of moles of tryp-
tophan per mole of protein) and Mtyr (the number of moles
of tyrosine per mole of protein), are found by solving the

two simultaneous equations:

NtrpllBlS + MtyrB‘oS

E 288

Ntrp5690 + Mt 1.1280

8280 = Y

and g are the extinctions of the unknown
s 230

protein solution at 288 nm and 280 nm. 5690 and 1280 are

here

VV E28
the known extinctions of tryptophan and tyrosine at 280 nm.
4815 and 585 are the known extinctions at 288 nm

Since A = E. x conc.(for a 1 cm cuvette)
.. A 1.: x 104’-

£280 - [Proteini a .19 x 10- M 7' 5.10 x 10‘ M—1
2 AgEm ... %.5 x 10-2 -

£1233 [Protein] .19 x 10- M r 2.27 x 10‘ M-1

.. 3 _ 4

Ntrp .. (gees/3.103 x 10 ) (gage/1.032 x 10 )
‘=(2.23 x 10‘/3.103 x 103) - (3.10 x 10‘/1.032 x 104)
='4.31

(4.815 x m").- Mtyr(3.

(3.85 x 102)

2
£288 =Ntrp 85 x 10 )

2.27 x 104 - 2.075 x 104

Mtyr
1.95 x 103 = Mtyr (3.85 x 102)

Mtyr = 5'07

158

159
Number average molecular weights for denatured gh-l DNA
and denatured gh-l DNA limit polymers

 

Specific activity of 32P-ATP=2.38 x 103cpm/lumole

A. denatured gh-l DNA

32 -
32 . P (cpm) incorporated
/umoles P incorporated azP-ATP specific activity

H

: 6 x 102 cpm
2.58 x 10 chmole

2.52 x 10-°/Amole 32P incorp.

moles of 911-1 DNA* __ weight of gh-lDNA (gins)
MW of nucleotide (gm/mole)

l

$430 x 10-° ms
3.5 x 10 gms/mole

- 8.6 x 10-2,/Amoles

#moles of gh-l DNA __. 8.6 x 10—2 moles gh-l DNA
.xxmoles 32P incorporated 2.52 x 10-

moles P incorp.

 

5.4 x 10‘
Therefore one 32p atom was incorporated at the 5' end of
polymers containing an average of 3.h x 10‘ nucleotides.
MW of denatured gh-l DNA ‘2 MW of nucleotide x no. of nucleotides

=.5.5 x 102 x 3.4 x 104

H

1.2 x 107

 

* moles of DNA are expressed as moles of nucleotide equiva-

lents

160

B. denatured gh-l DNA limit polymers

4.43 x 103 cpm
‘/Amole532P incorporated :3 2.5 x 10 cpm[/4mole

: 1.87 x lo-fiamoles 322 incorp.

 

‘28 x 10-° ms .
moles of gh-l DNA limit polymers 5.5 x 10 gms/mole

=8 x 10-2 ,umoles

/umoles of gh-l DNA limit polymers 2 8 x 10-2
,xxmoles 32F incorporated 1.87 x 10-5

)I

4.27 x 103
Therefore one 32F atom was incorporated at the 5' end of
the polymers containing an average of 5.4 x 104 nucleo-
tides.
MW of denatured limit polymer ='MW of nucleotide x no. of
nucleotides
= 5.5 x 102 x 4.27 x 103
:= 1.49 x 10‘3

 

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