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This is to certify that the
thesis entitled

SP Lyase Recognition and Binding to DNA

presented by

Egidijus Zilinskas

has been accepted towards fulfillment
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SP LYASE RECOGNITION AND BINDING T0 DNA
By

Egidijus Zilinskas

AN ABSTRACT OF A THESIS
Submitted to
Michigan State University
in partial fiIlfillment of the requirements
for the degree of

MASTER OF SCIENCE
Department of Chemistry

2004

ABSTRACT

SP LYASE RECOGNITION AND BINDING T0 DNA

By

Egidijus Zilinskas

UV radiation is a mutagen and can trigger the formation of a series of lesions
within DNA. 5-Thyminyl-5,6-dihydrothymine (“spore photoproduct”) is generated in
Bacillus Spores upon irradiating with 254 nm UV light. Spore photoproduct lyase (SPL)
specifically targets the spore photoproduct and catalyzes its repair to two thyrnines. SPL
is a member of family of enzymes that involve [4Fe-4S] clusters and S-
adenosylmethionine (AdoMet) to generate catalytically essential radicals. Investigations
of the interactions between SPL and DNA/oligonucleotides that have adjacent thymines,
were carried out using agarose gel and gel-shift assays. The results suggest that SPL
binds to DNA. DNA repair was done by using 3H-labeled pUC18 DNA and SPL. Various
attempts to generate spore photoproduct from thymidine at different conditions and at

different UV wavelengths were also made.

ACKNOWLEDGEMENTS

I would like to thank my advisor, Prof. Joan B. Broderick for all the help and
guidance through my graduate studies. I also want to thank all the guys from the lab for
help and support (with Special thanks to Mbako). I’m especially gratefiil to Magdalena
for helping me out with NMR and the other stuff; Thalia, for the discussions about the
gel-shift assays (and not only); and Jeff for teaching me all the useful things in the lab.
And last, but not the least — I want to thank my parents, for all the emotional support and

help.

iii

TABLE OF CONTENTS

LIST OF SCHEMES ................................................................................ vi
LIST OF TABLES .................................................................................. vii
LIST OF FIGURES ................................................................................ viii
LIST OF ABBREVIATIONS ....................................................................... x
CHAPTER I

INTRODUCTION .................................................................................. 1
1.1 DNA and spore photoproduct ................................................................... 1
1.2 Small acid-soluble protein and spore photoproduct lyase ................................... 4
1.3 Adenosylmethionine-dependent iron-sulfur enzymes ....................................... 6
CHAPTER II

OVEREXPRESSION AND CHARACTERZITION OF SPORE
PHOTOPRODUCT LYASE ..................................................................... 14
11.1 Introduction ..................................................................................... 14
11.2 Experimental methods ......................................................................... 18
11.3 Results and Discussion ........................................................................ 21
CHAPTER III

GENERATION AND REPAIR OF ISOTOPICALLY LABELED SPORE
PHOTOPRODUCT ................................................................................ 24
111.] Introduction .................................................................................... 24
1112 Experimental methods ........................................................................ 26

iv

1113 Results and Discussion ....................................................................... 29

CHAPTER IV

SPORE PHOTOPRODUCT LYASE BINDING TO DNA AND

OLIGONUCLEOTIDES .......................................................................... 37
IV.1 Introduction .................................................................................... 37
IV.2 Experimental methods ........................................................................ 40
IV .3 Results and Discussion ....................................................................... 44
CHAPTER V

PHOTOSYNTHESIS OF SPORE PHOTOPRODUCT FROM THYMIDINE ...... 55
V.1 Introduction ..................................................................................... 55
V2 Experimental methods ......................................................................... 60
V3 Results and Discussion ........................................................................ 62
CHAPTER VI

CONCLUSIONS ................................................................................... 66
REFERENCES ...................................................................................... 68

LIST OF SCHEMES

Scheme 1.1 UV photoproducts, a) cyclobutane pyrimidine dimer,
b) pyrimidine-pyrimidone (6-4) photoproduct, c) spore photoproduct,

(5-thyminyl-5,6-dihydrothymine) ................................................ 2
Scheme 1.2 The repair of spore photoproduct by SPL to thymines ........................ 6
Scheme 1.3 Enzymatic reactions of the radical-SAM superfamily ......................... 7

Scheme [.4 The proposed mechanism of SP repair by spore photoproduct lyase ...... 12
Scheme 1.5 Modified reaction mechanism of SPL .......................................... 13
Scheme 11].] Tritium labeled C-6 spore photoproduct ....................................... 27
Scheme V.l Formation of various thymidine photoproducts under UV irradiation. . ...56
Scheme V.2 The UV-light induced formation of thymyl and thyminyl radicals... . . . . .57
Scheme V.3 Concerted mechanism for the formation of the hexadeuterated

5,6-dihydro 5-(ct-thymidyl) thymidine upon exposure to far-UV light

in frozen aqueous solutions ....................................................... 59
Scheme VA The energy levels of the lowest excited singlet states (SI) and lowest

triplet states (T1) of adenine (A), guanine (G), cytosine (C), thymine (T),
and acetophenone (¢Ac) ......................................................... 59

vi

 

Table 111.1

Table IV.1

Table IV.2

Table IV.3

Table IV.4

Table IV.5

Table IV.6

Table V.1

LIST OF TABLES

DNA bases and their elution times from HPLC .............................. 31

DNA and SPL binding conditions for agarose gel ............................. 47
Linear DNA and SPL binding conditions for agarose gel ................... 48
The binding conditions for gel-shift assay ..................................... 50
The binding conditions for gel-Shift assay ..................................... 51
The binding conditions for gel-Shift assay ..................................... 52
The binding conditions for gel-shift assay ..................................... 53
Various conditions for making SP from thymidine ........................... 64

vii

 

Figure 1.1
Figure 1.2

Figure 11.1

Figure 11.2

Figure 11.3

Figure 11.4

Figure 11.5
Figure 11.6
Figure 111.1
Figure 111.2

Figure 111.3

Figure 111.4

Figure 1V.1

Figure IV.2
Figure IV.3
Figure IVA
Figure 1V.5

Figure 1V.6

LIST OF FIGURES
Images in this thesis are presented in color.
Different conformations of DNA ................................................. 3
Sequence of SPL and the radical SAM superfamily enzymes ................ 9

Titration of SPL with sodium dithionite, monitored by UV-visible
Spectroscopy ........................................................................ 15

Temperature dependent EPR signal of the as isolated SPL (137uM)

in SOmM Hepes, 300mM NaCl, 10mM BME, lSOmM imidazole,

5% glycerol, pH=7.2 .............................................................. 15
Mdssbauer spectrum of anaerobically isolated 57Fe-em'iched SPL. . . . . ....16

Temperature dependent EPR spectrum SP lyase-Hi56 after the
reduction with photoreduced 5-deazariboflavin .............................. 17

Chromatogram of purification of SPL .......................................... 22

SDS-PAGE analysis of SPL fractions off the Ni-NTA affinity column...23

Thymine dimer production depending on conditions ......................... 32
SP repair assay ..................................................................... 34
Thymine dimers before and after 2 hours of re-irradiation at

254 nm wavelength ............................................................... 35
Thymine dimers before and after 4 hours of re-irradiation

at 254 nm wavelength ............................................................ 36
One of the regions of sequence homology between

SPL and DNA photolyases ...................................................... 38
Agarose gel of circular and linear pUC18 DNA .............................. 47
Agarose gel of linear pUC18 DNA at 4 0C .................................... 48
Gel-shift assay of the 32P-labeled 35-bp oligonucleotide .................... 50
Gel-Shift assay of the 32P-labeled 35-bp oligonucleotide .................... 51
Gel-shifi assay of the 32P-labeled 94-bp oligonucleotide .................... 52

viii

Figure IV.7 Gel-Shift assay of the 32P-labeled 94—bp oligonucleotide .................... 53

Figure V.1 1H NMR spectrum of thymidine and its dimers (in D20) .................... 65

ix

LIST OF ABBREVIATIONS

5’-dAdo .......................................................................... 5’-deoxyadenosine
Adomet ................................................................... S-adenosyl-L-methionine
ATP ........................................................................... adenosine triphosphate
APS ............................................................................ ammonium persulfate
B-ME .............................................................................. ,B-mercaptoethanol
BSA ............................................................................. bovin serum albumin
EDTA ............................... (Ethylendinitrilo)tetra-acetic acid disodium salt dihydrate
HPLC ................................................... hi gh-performance liquid chromatography
DTT ........................................................................................ dithiotreitol
E. coli ................................................................................. Escherichia coli
EPR ............................................................... electron paramagnetic resonance
Hepes .................................. N-(2-hydroxyethyl)piperazine-N’-2-ethanesulfonic acid
IPTG ......................................................... isopropyl-fl-D-thiogalactopyranoside
LAM ........................................................................ lysine 2,3 —aminomutase
LB ........................................................................................ Luria-Bertani
NMR .................................................................... nuclear magnetic resonance
MOPS .................................................... 3-(N-morpholino) propanesulfonic acid
PFL ............................................................................ pyruvate formate-lyase
PFL-AE ................................................ pyruvate formate-lyase‘ activating enzyme
PMSF ................................................................ phenyhnethylsulfonyl fluoride
SDS-PAGE ....................... sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SP ................................................................................. Spore photoproduct

SPL ........................................................................ spore photoproduct lyase
SspC ....................................................................... small acid soluble protein
T4 PNK .................................................................... T4 polynucleotide kinase
TEMED ................................................ N,N,N’,N’-Tetra-Methyl-ethylendiamine
TFA ............................................................................... trifluoro acetic acid
Tris ............................................................. tris(hydroxymethyl)aminomethane

xi

CHAPTER I

INTRODUCTION

11 DNA and Spore photoproduct
The chemical stability of DNA is one of the prerequisites of life. Even though
DNA is a very stable molecule (more stable than proteins and RNA), this stability is not
sufficient for its role as the long-term carrier of genetic information. Alterations in the
structure of DNA occur frequently and DNA damage can be classified in two categories
(1):
1) Intrinsic DNA damage (oxidative damage, deamination of bases, or mismatches
during DNA synthesis)
2) Environmental DNA damage (chemical, for example from cross-linking agents;

physical, such as ionizing and UV radiation)

UV radiation is a mutagen and it is the most harmful component of the solar radiation
spectrum. UV induces the formation of a series of lesions within DNA. In the UV-C
(100-290 nm) and UV-B (290-320 nm) range, damage is triggered by the direct
absorption of the incident light by DNA bases (1, 2). Reactions of the resulting excited
species lead mostly to the dimerization of pyrimidine bases thymine (T) and cytosine (C).
The major DNA photoproducts of UV irradiation are cyclobutane pyrimidine dimers (TT,
CT, and CC dimers) and 6,4-photoproducts, while S-thyminyl-S,6-dihydrothymine is a
minor product (Scheme I.1)(3, 4). UV-A (320-400 nm) light also has cytotoxic and

mutagenic features, however to a smaller extent than UV-B radiation. DNA is not a

chromOphore for a UV-A radiation with the exception of photons around 360 nm. It is
likely that UV-A photons are absorbed by the unidentified endogenous photosensitizers
that may damage DNA through photooxidation reactions (4). Different cells, however,
can respond differently to UV irradiation. The major UV photoproduct formed in DNA of
vegetative cells is a cis,syn-cyclobutane-type thymine dimer, while in spores the major
photoproduct, generated by 254 nm UV irradiation, is 5-thyminyl-5,6-dihydrothymine

(“spore photoproduct”) (5-7).

 

 

o .H H
\ f—N \ O)——N’
)Igar—N O sugar—N 0
ho hate H CH / , E
P 5P\ O N CH 3 phospha\te oH ,H CH2
sugar—N‘ H OH sugar—N) N\? O
H CH3 H H CH3
A B C

Scheme 1.1 a) Cyclobutane pyrimidine dimer, b) pyrimidine-pyrimidone (6-4)
photoproduct, c) spore photoproduct, (5-thyminy1-5,6-dihydrothyrnine)

Dormant Spores of the various Bacillus and Clostridium species, including
Bacillus subtilis, are 5 to 20 times more resistant than vegetative cells to UV radiation
(8). The Spore resistance is due to:

a) the UV photochemistry of DNA within spores which generates few if

any cyclobutane dimers, but significant quantities of SP, and

b) DNA repair, and particularly SP-specific repair, during spore

germination.

The novel UV photochemistry has been proposed to be a result of DNA being in the A
conformation rather than the B conformation in bacterial spores (Figure I.1)(9). However
other studies have shown that SP formation was not necessarily dependent on DNA
conformation, but rather on DNA hydration, with increasing SP and decreasing
cyclobutane dimers observed as DNA hydration decreased (10). Since the levels of water
in the spore core are low (1 1), the explanation is that the reduced hydration in the core
causes a conformational change in Spore DNA resulting in its novel UV photochemistry
(9). Spore DNA was suggested to be in an A-like conformation, but during sporulation
the spore DNA photochemistry changes well before full spore core dehydration takes
place, suggesting that DNA hydration is not the only factor. In addition, while UV

irradiation of poorly hydrated DNA in vitro gives SP, significant amount of cyclobutane

 

A DNA 2 DNA

     

 

 

 

Figure 1.1 Different conformations of DNA

dimers is also generated (9). Different hydration levels were tested in vitro, but
cyclobutane pyrimidine dimer formation was still Significant, and so hydration levels
alone do not appear to explain the unique photochemistry observed in bacterial Spores

(10).

1.2. Small acid-soluble proteins and spore photoproduct lyase

The other reason for unique spore photochemistry could be the presence of small
acid-soluble proteins (SASP), which appear 3-4 hr after the onset of sporulation in
bacteria and constitute up to 20% of the protein in mature spores (8). These proteins
range from 60 to 73 amino acids (5-7 kDa) and belong to a multi-gene family that is
highly conserved and found in spore-forming bacteria such as Bacillus and Clostridia.
These small proteins exhibit a very high degree of amino acid sequence homology both
within and across Bacillus species. However, these proteins exhibit no obvious homology
to any other proteins in available databases. The CUB-type SASP are encoded by a
multigene family, with individual members (termed ssp genes) scattered widely on the
chromosome as small (200 bp to 300 bp) monocistronic transcription units; the ssp genes
are expressed in parallel only in the developing spore midway to sporulation (8). During
spore germination the a/B-type SASP are rapidly degraded to amino acids, with their
degradation initiated by a novel endoprotease termed the germination protease (GPR),
which recognizes and cleaves within a pentapeptide sequence conserved in all cz/B-type
SASP (8). The SASP’S presence has been correlated with the marked resistance of
Bacillus to UV radiation, as deletion mutants of the SASP’S lose their UV resistance (12).

It also has been Shown that SASP’S are associated with the Spore chromosomal DNA in

viva. It has been proposed that binding of the SASP’S to DNA promotes a structural
change (13) and a change in the level of hydration of the DNA that results in formation of
SP rather than cyclobutane thymine dimers (l4). SASP’S have been Shown to be largely
random coil in the absence of DNA, but to fold into a Int-helical structure when bound to
DNA (15). Spectroscopic evidence also suggests that SASP binding to DNA converts the
DNA into what can best be described as A-from (Figure I.1)(13). However, the structure
of the SASP-DNA complex has not been determined and it is not known if TT sequences
have an impact on conformation that may help explain the formation of SP rather that
other photoproducts.

Repair of UV-induced damage helps to prevent of a number of adverse conditions
such as a melanoma. Pyrimidine dimers such as SP damage the cells by blocking
replication and transcription, which can result mutations if transcription does proceed
past the region of the dimer. Repair of these dimers is therefore important in order to
avoid mutations and is the key to UV resistance. Pyrimidine dimers can be excised and
replaced, but the only example of direct pyrimidine dimer conversion to the starting
material is the photoreactivation catalyzed by DNA photolyase. However, this reaction
has been Shown to be absent in many Species, including Bacillus, suggesting that
alternate means of pyrimidine dimer repair might be found (16).

The enzyme Spore photoproduct lyase (SPL) iS the first identified pyrimidine
dimer lyase that does not require photoactivation to repair spore damaged DNA (17).
Together with the SASP’S, SPL confers on Bacillus spores their unusual resistance to UV
radiation (18). Bacterial spores can survive harsh conditions for extended periods of time

(11). During dormancy these spores are unable to repair damage to their DNA from UV

exposure, which is caused by accumulation of SP. Spores can, however, undergo DNA
repair in the very early germination cycle through two repair mechanisms (11, 16):

o the nucleotide excision repair pathway (NER), and

o the reversal of SP to thymine catalyzed by the enzyme spore photoproduct

lyase.

NER detects and removes lesions from the DNA including SP and cyclobutane-type
pyrimidine dimers (1). SP lyase specifically targets the SP and cleaves into two thymines

by an unknown but light-independent mechanism (19) (Scheme 1.2).

'O'Punu’

l

sugar——-—N
O
sugar—N

‘YZI
\

3’

7%
\/
I. l

sugar-—
/

gar—N

X

phosphat\

——>
be

UV Light

MIIII

0
Scheme 1.2 The repair of spore photoproduct by SPL to thymines

1.3 Adenosylmethionine-dependent iron-sulfur enzymes

Iron-sulfiir clusters are widespread in biological systems and participate in
electron transport, as well as mediating redox catalysis and non-redox catalysis (20).
Aconitase is one of the most studied enzymes that mediate non-redox catalysis, in which
a [4Fe-48]2+ cluster serves as a Lewis acid to catalyze the interconversion between citrate
and isocitrate (20). F e-S clusters are also involved in regulation of gene expression, for
example in the iron-responsive protein (IRP) and in SoxR, which respond to changes in
levels of iron and superoxide, respectively (20). Another role of F e-S clusters is that some

enzymes use Fe-S cluster and AdoMet to initiate radical catalysis. This Fe—S cluster-

mediated radical catalysis includes glycyl radical formation, rearrangement reactions,

cofactor biosynthesis, and repair of DNA damage (Scheme 1.3)(21, 22).

Activating Enzyme for Pyruvate F ormate Lyase

0 CH3
0 O H 0' “N NH
COOH
Biotin Synthase S
o o T
HN/JL\NH -——*> HN’JL\NH
WCOOH WCOOH
H3O H20
\SX

Activating Enzyme for Benzylsuccinate Synthase 'OZC

+ I --* c

Lipoic Acid Synthase

OH S-S
\/\/\/\n/ . OH
O
O
Lysine 2,3-aminomutase
\\N H3+ \\+NH3
W W

Scheme 1.3 Enzymatic reactions of the Radical-SAM superfamily

coz‘

More that 600 related enzymes that involve AdoMet-derived radical biochemistry have
been recently identified (21). The activating enzymes include two E. coli enzymes that
generate catalytically essential radicals on enzymes required during anaerobiosis, the
anaerobic ribonucleotide reductase-activating enzyme (aRNR-AE) and the pyruvate
formate-lyase activating enzyme (PFL-AE)(23). Lysine 2,3-aminomutase (LAM)
catalyzes a rearrangement reaction analogous to those catalyzed by adenosylcobalamin-
dependent enzymes, except that no cobalamin cofactor is required (24). Biotin synthase
and lipoic acid synthase catalyze sulfur insertion reactions (25, 26), presumably by a
radical mechanism (27, 28). For both PFL-AE and LAM, label transfer studies have
provided evidence for an adenosyl radical intermediate derived from the required
AdoMet cofactor (22). The information available on these enzymes suggests that all
catalyze radical chemistry mediated by S-adenosylmethionine, although the mechanistic
details have yet to elucidated (20).

Previous work suggested that SP lyase utilized S-adenosylmethionine (AdoMet)
and contained an iron-sulfur cluster, suggesting that it was a member of the Fe—S/AdoMet
family of enzymes (29). As shown in Figure 1.2, the Fe-S/AdoMet enzymes have in
common a three-cysteine motif, and these three cysteines have been Shown for several of
the enzymes to be responsible for coordination of the catalytically essential iron-sulfur
cluster (20, 22). SP lyase also contains three cysteines in the CX3CX2C motif unique to
the Fe-S/AdoMet enzymes, and significant sequence homology to the Fe-S/AdoMet

enzymes is observed between residues 80 and 115 of SP lyase (20).

 

 

OSP Lyase 86 IPFATGCMGHCHYCYLQTT 104
CPFL-AE 24 ITFFQGCLMRCLYCHNRDT 42
0aRNR-AE 20 VLFVTGCLHKCEGCYNRST 38
oBiotin Syn 47 SIKTGACPQDCKYCPQTSR 65
oLipoate Syn 48 MILGAICTRRCPFCDVAHG 66
OLAM 132 LLITDMCSMYCRHCTRRRF 150

 

 

Figure 1.2 Sequence of SPL and the radical SAM superfamily enzymes. SP
Lyase, spore photoproduct lyase; PFL -AE, pyruvate formate-lyase activating enzyme;
aRNR-AE, anaerobic ribonucleotide reductase activating enzyme; Biotin syn, biotin

synthase; Lipoate syn, lipoate synthase; LAM, lysine 2,3-aminomutase.

Purified SP lyase has been Shown to contain iron and acid-labile sulfide, and the
UV-visible spectrum is suggestive of the presence of an iron-sulfur cluster (29, 30).
Addition of AdoMet to the in vitro activity assay was found to stimulate SP repair
activity of SP lyase (29). This and other early studies of SP lyase, however, were
hampered by the inability to isolate active enzyme in sufficient quantities for
physicochemical studies (30). The limited spectroscopic and mechanistic studies that
have been done on SP lyase were therefore done on inactive enzyme (29, 30), leaving
open numerous questions, including the relevance of the purported F e/S cluster and the
AdoMet dependence.

SP lyase also Shares some sequence homology with the members of the DNA
photolyase/(6-4) photolyase/blue light photoreceptor protein family (18). The conserved
residues are in the general region believed to be involved in substrate binding by DNA

photolyase. Both SP lyase and photolyase perform the same type of chemistry, which is

the cleavage of nucleotide dimers to monomers, however there are some major
differences:
1) the DNA photolyases, but not SP lyase, are activated by visible light (31),
2) the DNA photolyases contain a flavin (FAD) and either folate or a deazaflavin
(32), while SP lyase contains an F e-S cluster (29), and
3) the DNA photolyases cleave cyclobutane pyrimidine dimers to monomers (33)

while SP lyase cleaves methylene-bridged thymine dimers to monomers (18).

The commonly accepted mechanism for photorepair by DNA photolyase involves dimer
splitting as a consequence of a Single electron transfer fiom the enzyme to the dimer, a
mechanism recently supported by quantum chemical calculations (34). The functional
similarity between SP lyase and the F e-S/AdoMet enzymes implies that cleavage of the
SP dimer also occurs through an iron-sulfur cluster-mediated radical mechanism.

The putative involvement of an F e-S cluster in DNA repair suggests that the
cluster could serve as a Spectroscopic handle for detailed mechanistic studies of thymine
dimer repair. The preliminary observations on SP lyase point to a novel mechanism of
repair of UV-induced DNA damage, which involves a Fe-S cluster and AdoMet (29).
This mechanism was initially proposed by Mehl and Begley (35), and further suppOIted
by Check and Broderick (36). The latter workers Showed that 5’dAdo radical is involved
in the SP lyase enzymatic mechanism (Scheme 1.4). During the repair 3H label was
transferred from C-6-tritiated Spore photoproduct to AdoMet (36). This label transfer
suggests that SP repair is initiated by 06 H atom abstraction, with the resulting substrate

radical presumably undergoing B-SCission to provide the monomeric products (36).

10

AdoMet also acts as a catalytic cofactor during repair to reversibly generate the putative
adenosyl radical intermediate (Scheme 1.4)(36). The quantum chemical calculations,
which employed hybrid Hartree-Fock/density functional theory, proposed that an inter-
thymine hydrogen atom transfer step takes place before the back-transfer of the hydrogen
atom from the adenosine cofactor (37). The last step is shown to be the rate-determining
step in the reactions (Scheme 1.5)(37).

It is known that SPL is one of the radical SAM-superfamily enzymes and it
repairs SP (29). It was also shown that SPL is similar to DNA photolyases (18), but there
is little knowledge on how SPL recognizes damaged DNA and how much of SP can be
repaired, how repair depends on time and other factors. In our research we focused on
creating SP in pUC18 DNA and doing SP repair assays. We also tested how SPL binds
on pUC18 DNA and short nucleotides. More research, however, has to be done to
investigate if SPL binding is specific and how SPL recognizes thymine-thymine dimer
and/or SP. Various attempts to generate Spore photoproduct fi'om thymidine at different

conditions and at different UV wavelengths were also made.

11

DNA

 

Scheme 1.4 The proposed mechanism of SP repair by spore photoproduct lyase (36)

12

|

—— o
phosphate/sugar g>_$>= —>phosphate/sugar—g>_rg
Hug—g Him...“
sugar—gl>__u o sugar—N; u
Ado-DHz Ado-CH3
l a...
/sugar——:;__—_g§=o

phosphate

IZ

l
l

/sugar———N
u ar-——N

g N

H

phosphate
..~/—§:;——o
ugar N
H o
Ado-CH2 Ado-CH3
sugar—N>_/—__§=O

0

phosphate/ 0 if:
\ r;
U r—-—-N O

93 2—2

Ado-CH3

Scheme 1.5 Modified Reaction Mechanism of SPL (Adapted from Ref. (3 7))

13

CHAPTER II

OVEREXPRESSION AND CHARACTERIZATION OF SPORE
PHOTOPRODUCT LYASE

11.1 Introduction

The enzyme SP lyase is the first identified non-photoactivatable pyrimidine dimer
lyase, and, together with the SASP’S, is responsible for Bacillus spores unusual resistance
to UV radiation. The SP lyase was previously characterized using both analytical and
Spectroscopic methods (30). It was found the enzyme contains iron (1.8 mol/mol protein)
and acid-labile sulfide ( 1.8 mol/mol protein), consistent with the presence of an iron-
sulfur cluster. The UV-visible Spectrum of the purified protein exhibits a broad shoulder
with maxima at 410 nm (4.75 mM’lcm") and 450 nm (4.03 mM'lcm") (FigureII.l).

The electron paramagnetic resonance (EPR) spectrum of the isolated enzyme
exhibits a sharp nearly isotropic resonance that has g values, anisotropy, and relaxation
properties consistent with its assignment to a [3Fe-4S]1+ cluster (Figure 11.2) (30). This
EPR signal typically accounts for 30-40% of the iron in the sample, and therefore a
Significant proportion of the iron is present in an EPR-silent state. The data from
Mossbauer Spectroscopy confirms the presence of approximately 40% [3Fe-4S]1+ cluster,

and the remaining iron is present as a [2Fe-ZS]2+ cluster (Figure 11.3) (30).

14

2.0 -

 

— As Isolated
— Reduced w/ dithionite

 

Absorbance

 

 

' l ' l ' I ' *1
300 400 500 600 700 800
Wavelength (nm)

Figure 11.1 Titration of SPL with sodium dithionite, monitored by UV-visible
spectroscopy. The decrease in the visible absorption is consistent with reduction of the

cluster by sodium dithionite (30).

20K

E’R Intensity

30K

35
40K

 

3000 3100 3200 3300 3400 3500 3600 3700 3800
Field (Gauss)
Figure 11.2 Temperature dependent EPR signal of the as isolated SPL (l37uM) in 50mM
Hepes, 300mM NaCl, 10mM BME, lSOmM imidazole, 5% glycerol, pH=7.2 (30).

 

leTlllllllrlllll

-----------------

7f .

0.4 I-

 

 

 

ABSORPTION (96)
5'"
g

0.6 )-

 

 

E§ Er

 

 

11111114111111111
-8 «l 0 4 8

VELOCITY (mm s)

 

Figure 11.3 Messbauer spectrum of anaerobically isolated 57Fe-enriched SPL in
50mM Hepes, 300mM NaCl, 10mM BME, ISOmM imidazole, 5% glycerol, pH=7.2. The
spectrum was recorded at 4.2 K in a magnetic field of 50 mT applied parallel to the y-
rays. The solid line plotted above the data is a theoretical simulation of a [2Fe-ZS]2+
cluster. This component accounts for 47% of the total Fe absorption. The dashed line is a
theoretical Simulation of a [3Fe-4S]1+ cluster. The [3F e-4S]1+ signal accounts for 43% of
total intensity. The solid line overlaid with the experimental data is the sum of the
contributions of the [2Fe-ZS]2+ and [3F e-4S]l+ clusters (30).

SP lyase can be reduced by titration with sodium dithionite, as is evidenced by the
decrease in visible absorption that is characteristic of reducing an iron-sulfur cluster
(Figure 11.1) (30). An alternative method of reducing SP lyase is by photoreduction in the

presence of 5- deazariboflavin. The reduction of SP lyase results in a dramatic change in

16

 

 

EPR Intensity

 

 

 

 

 

 

 

 

2800 3000 3200 3400 3600 3800 4000
Field (Gauss)

Figure 11.4 Temperature dependent EPR spectrum SP lyase-His6 after the
reduction with photoreduced 5-deazariboflavin. The axial signal with g values of 2.04,
1.94 are characteristic of a [4Fe-4S]'+ cluster. Spin' quantitation reveals that this signal

accounts for approximately 41% of the total iron (30).

the EPR spectral properties (Figure 11.4). Rather that the fairly intense, nearly isotropic
Signal observed for the as-isolated enzyme (Figure 11.2), the reduced enzyme has a weak,
nearly axial Signal that is characteristic of a [4Fe-4S]1+ cluster(Figure 11.4). Integration of
this Signal shows that it accounts for approximately 40% of the total iron in the protein.
The remaining iron is present in an EPR-silent state which is believed to be the [4Fe-
4S]2+ state based on preliminary Mdssbauer studies (30). Therefore reduction appears to
convert the [3Fe-4S]1+ and [2Fe-ZS]2+ clusters to [4Fe-4S]2+/1+ clusters (30). Such [3Fe-
4S]1+—>[4Fe-4S]2+”+ reductive cluster conversions are believed to result from

“cannibalization” of the [3Fe-4S] 2”” clusters to form the [4Fe-4S]1+ clusters, which are

17

more thermodynamically stable under reducing conditions (30). Whether these cluster
conversions have any physiological relevance for any of the Fe-S/AdoMet enzymes has

not be determined yet.

11.2. Experimental methods
11.2.1. Growth and expression of SPL

The gene for spore photoproduct lyase splB was previously cloned into the pET-
l4b (Novagen) vector in order to attach an N-terminal 6 histidine (H186) tag generating
SP lyase-His; (36). The plasmid pEt-14b-SPL17 was purified and was transformed into
the bacterial competent cells, TUNER(DE3)pLysS Escherichia coli (Stratagene). The
procedure for this transformation involves the following method. TUNER(DE3)pLysS
competent cells (50 ul) were thawed on ice and 1 mL of a pET14b-SPL17 plasmid
solution was added. Cells were incubated on ice for 10 minutes and then immersed in a
42 °C water bath for 30 seconds followed by incubation for 2 minutes on ice. SOC media
(100 pl) was added and the cells are Shaken at 37 °C for 30 minutes. Then cells were
removed fi'om the Shaker and plated on agar plates containing Luria Base Broth media
and ampicillin (50 ug/mL). The plates were incubated overnight for 16 hours at 37 0C.
Small white colonies were seen after incubation.

A Single colony was used to inoculate a 50 mL solution of LB media containing
50 ug/mL ampicillin and grown overnight for 16 hours at 37 0C in the shaker at 250rpm.
This overnight culture was used to inoculate 6 Bellco flasks containing a minimal media
(950 mL per flask). Minimal media included (per 6 L): 66 g of casamino acids, 50.4 g

MOPS, 17.32 g NaCl, 4.6 g Tricine, 3.06 NH4C1, 4.6 g KOH and 5.8 L MQ Water, 80

18

mL “0” solution, 80 mL “P” solution (1M KII3PO4), 40 mL “S” solution (276 mM
K2804), 160 mL of glucose (40 g in 200 mL), 200 mL 0.1 M CaClz. The “0” solution
consists of 0.1 g FeClszZO, 10 mL conc. HCl, lmL “T” solution, 2.68 g MgC12x6HzO
in 50 mL of water. The “T” solutions consists 8 mL conc. HCl, 18.4 mg CaClszzO, 64
mg H3303, 40 mg MnC12x4H20, 18 mg CoClzx6H20, 4 mg CuClzx2HzO, 340 mg ZnClz,
605 mg NazMoO4x2H20 in 100 mL with MQ water. Ampicillin 6 mL of 50 mg/mL and
10 mg each of pyrodoxine, riboflavin, niacinamide, biotin, vitamin B12, pantothenic acid,
thioctic acid, folic acid, thiamine (vitamin B1) were added to 6 L of culture right before
inoculation. The flasks were placed in a New Brunswick Scientific shaker at 175 rpm (37
°C) and allowed to grow to an optical density of 0.6 at 600 nm. After the optical density
at 600 nm reached 0.6, 1 mL of isopropyl-fl-D-thiogalactopyranoside (IPTG) was added
with 300mg of Fe(NH4)2(SO4)2 to each flask. The cell culture is grown for additional 3
hours before harvesting by centrifugation at 10,816xg (8000 rpm, Sorvall GS3 rotor). The
supernatant was decanted and the cells stored at —80 °C. A typical yield of cells is 10-20 g

for 6 L of media.

11.2.3 Purification of SPL

SPL was purified from these cultured Tuner(DE3)pLySS cells transformed with
the vector pET-14b-SPL17. All steps of purification were carried out in a Single day
under anaerobic conditions at 4 0C in a Coy anaerobic chamber (Coy laboratories, Grass
Lake, MI). All buffers were degassed prior to use by purging with N2 for over 1 hour or
by repeated pump/purge cycles on the Schlenk line. Approximately 10 g of the cells were

brought into the anaerobic chamber and resuspended in 20 mL of a lysis buffer

19

containing 50 mM Hepes, 200 mM NaCl, 1% Triton X-100, 5% glycerol, 10 mM MgC12,
5 mM BME, 1 mM PMSF, 1mg lysozyme and trace amounts of (approximately 0.1 mg
each) Rnase A and Dnase 1 (pH 7.5). The cell suspension was agitated for one our and
centrifuged at 27,000 rpm (rotor $834) for 30 minutes at 4 °C. A resulting greenish crude
extract was decanted and loaded onto Ni-NTA affinity column (1.6 x 10 cm, Qiagen)
equilibrated with Buffer A (50 mM Hepes, 300 mM NaCl, 5% Glycerol, 5 mM BME,
10mM imidazole, pH 7.5). After loading, the column was washed with 40mL of Buffer A
followed by a linear gradient from Buffer A to Buffer B (50 mM Hepes, 300 mM NaCl,
5% Glycerol, 5 mM BME, 250 mM imidazol, pH 7.5).

Aliquots from several fractions were suspended in SDS-PAGE sample buffer,
boiled, and centrifuged and an aliquot of the supernatant was loaded on SDS-PAGE gel
(12% Tris-HCl, BioRad). Fractions that were judged to be >95% pure were pooled. Later
the pooled protein was concentrated using an Amicon concentrator with a PM10
membrane. Standard Bradford assay techniques (Sec. 11.2.4) were employed to determine
the concentration of the pooled protein and a maximum concentration of ~10mg/mL of
protein was obtained before Significant precipitation of protein occurred. A typical yield

of protein is ~10 mg per 6L of culture.

11.2.4 Protein assays
Routine determinations of protein concentration were determined by the method

of Bradford (3 8), with bovine serum albumin is a standard.

11.2.5 Iron assays

Iron assays were carried out by using the method of Beinert (39).

20

1.2.6 Results and Discussion

In order to do SP repair assay, SPL binding assays and other experiments, first we
had to overexpress and purify SPL. SPL was purified from Tuner(DE3)pLysS cells
harboring the pET-14b-SPL17 plasmid. Inducing at an earlier log phase (ODGOO of ~0.6)
and supplementing the medium with iron after induction with IPTG contributed to better
overexpression and iron inclusion of SPL. The cells were lysed by an enzymatic
procedure.

SPL was purified by passing through Ni-NTA affinity column under strictly
anaerobic conditions (Figure 11.5) in the presence of DTT and pooling fractions based not
only on SDS-PAGE analysis (Figure 11.6), which Shows Sharp bands at ~42 kDa size
(Lanes 4-8), but also on the UV absorbance at 426 nm, which shows ligand-to-metal
charge transfer for iron-sulfur cluster, and 280 nm, which Shows the absorbance of
nucleic acids (Figure 11.5).

The purification of SPL resulted in the isolation of a 42 kDa protein that is about
95% pure according to SDS-PAGE results. The color of the protein was reddish-brown
and would change to clear when left exposed to oxygen for a period of time. The protein
was also sensitive to temperature and would precipitate rather quickly if left to sit at room
temperature. The typical yield of protein that could be purified from 6 L growth media
was 10 mg. The protein could be concentrated to a maximum of 150 uM, after which
precipitation would occur. The SPL contains 1.8 mol Fe/mol protein and a stoichiometric

amount of acid-labile sulfide.

21

 

1100
1000 4r "
900 v _-

 

 

700 < -
— A280

— A426
' -— hidazoie conc.

Au

 

 

 

4ooe-e- - -
300
200~ - r- -
100— , .

 

 

 

 

 

 

mL

 

 

 

Figure 11.5 Chromatogram of purification of SPL. Elution profile for the Ni-NTA
Affinity column (Qiagen, 1.5 x 25 cm), gradient from Buffer A to Buffer B over 200 mL,
flow rate 1 mL/min. SPL eluted through last half of the gradient. The peak at 70-80 mL is
the other proteins and a broad band at ~140-l70 is SPL.

22

45 kDa ‘M

Figure 11.6 SDS-PAGE analysis of SPL fractions off the Ni-NTA Affinity coltunn.
Lane 1, molecular marker (kDa); lanes 2-8, fractions 19-29 (odd fractions only).

Fractions 21-27 were pooled.

23

CHAPTER III
GENERATION AND REPAIR OF ISOTOPICALLY LABELED SPORE
PHOTOPRODUCT

111.1 Introduction

The different photochemistry between vegetative cells and spores is due to the
change of the conformation of DNA. This conformational change might be triggered by
environmental conditions and/or by the presence of small acid-soluble proteins. In the
early papers it was shown that DNA conformation changes when relative humidity is
lowered below 65% (10). At these conditions the DNA is disordered and a loss of base
stacking leads to a reduction of cyclobutane dimer formation and increase in SP
formation. But the enhancement in the yield of Spore photoproduct as the relative
humidity iS reduced cannot be explained simply by formation of disordered or unstacked
DNA, because no SP is observed upon irradiation of highly denatured DNA in solution
(10). It was also proposed, that SP formation depends on DNA conformation (5). It was
shown that in ethanolic solution native T7 DNA can undergo conformational transitions
from the B conformation (0% ethanol) to the C-like (60% w/w ethanol) and the A (80%
w/w ethanol) conformations (9). The formation of SP reaches a maximum when the
concentration of ethanol is 80% in either native or denatured DNA, while cyclobutyl
dipyrimidines are still the major class of photoproducts formed (9). It was assumed that
the lowered water activity itself, at high ethanol concentration, rather than the
conformational changes such an environment induces in DNA, is the critical factor in

formation of SP.

24

It was later discovered, however, that DNA conformational changes in spores are
due to the presence of small acid-soluble proteins (SASP)(15) and that SASP plays a
major role in formation of spore photoproduct (40, 41). SASPS bind to and saturate Spore
DNA, causing structure perturbations in the DNA such that the structure is more A-like
(42). It is believed that these structural perturbations induced by SASPS result in
formation of SP, rather than cyclobutane pyrimidine dimers, upon UV irradiation (41).
Although the presence of ct/B-type SASP largely explains the production of SP by 254
nm UV radiation, it does not explain spore UV resistance (15). When UV irradiation was
applied to [3H] thymidine-labeled DNA, the major photoproduct was cis,syn cyclobutane
dimer with smaller amounts of two other cyclobutane-type dimers, CT and trans,syn
cyclobutane dimer. In contrast, irradiation of a 5:1 complex (wt/wt) of SspC with DNA in
liquid produced very little pyrimidine dimer but a significant amount of SP (14). When
the complexes with increasing SspC/DNA ratio were used, production of cis,syn
cyclobutane dimer fell while production of SP increased. The SspC/DNA interaction in
the dry DNA films also appeared to saturate at an SspC/DNA weight ratio of 4-5:1 and at
this ratio cis,syn cyclobutane dimer formation was almost completely abolished (14).
Photoreactivity of a/B-type SspC/DNA complexes in vitro is similar to that in the Spore
in the first minutes, when the spores go through the period of transiently increased UV

resistance (14).

25

111.2 Experimental methods
111.2.1 Materials

AdoMet was purchased from Sigma-Aldrich. [methyl-3 H] thymidine
(0.74mBq/mL) was purchased from Amersham Pharmacia. pUC18 DNA was previously
made in our laboratory. High flash point cocktail Safety-Solve for scintillation counting

was purchased from Research Products International.

111.2.2 Generation of Spore Photoproduct

The plasmid DNA from pUC18 was transformed as described previously (Chapter
11.2.1) into NovaBlue E. coli (Novagen). A single colony of these bacteria was placed in
LB medium containing 50 ug/mL ampicillin, 0.45 mM deoxyadenosine, and 10 pM
[methyl-3H] thymidine and grown overnight for 16 hours at 37 °C in the shaker at 250
rpm. This process is intended to label the thymine bases in the DNA backbone at the
position Shown in Figure 12. The DNA from grown cells was extracted using a Promega
Wizard Mini-Prep Kit. Specific activity of the purified DNA was about 1.5x107
cpm/umol.

DNA (100, 0.186 uM) was placed on SaranWrap and left overnight to dry. The
following day the SaranWrap was placed over saturated ZnClz solution and left for 5-7
days to equilibrate. The tritium labeled DNA was then irradiated with ultraviolet light
(UVGL-25 lamp, 2x4 W, UVP) at 254 nm for 5 hours, to generate the tritiated spore

photoproduct (Scheme 111.1).

26

O

/sugar-—-N:‘_
phosphate H CH 3 +— Tritium labelled
\ HIW)L_; methyl group
sugar—N O
/ 02 ii

Scheme 111 1 Tritium labeled C-6 Spore photoproduct

”Winn"

IAZWI

Another set of the experiments included irradiation of H labeled DNA In ethanol

solution DNA (100 uL) was added to 100 uL of water and 800 uL of ethanol (total —
80%) The resulting solution was placed on a watch glass and irradiated at 254 nm

(UV GL 25 2x4 W, UVP) for 1, 2, and 5 hours while stirring With the magnetic stir bar
The third type of experiment Involved making a DNA/ethanol dry film. DNA (50

mL 0 186 M) was added to 450 uL of ethanol and placed on a watch glass and dried
ovemrght The dry film was then irradiated at 254 nm (UV GL-25, 2x4 W, UVP) for 5

hours and resuspended in 100 pL of water
111 2 3 Spore Photoproduct Repair Assays
SP lyase activity was determined in the following manner uSIng modified versron
of the assay developed by Nrcholson and coworkers. All work was carried out under

anaerobic conditions In an Mbraun glove box and the solutions were prepared InSIde the

27

glove box. The oxygen level was <1 ppm. Assay experiments were set up in parallel with
one containing SP lyase (10 ug) and the other containing no enzyme. A reaction mix of
200 uL was created containing 25 mM Tris-acetate pH 7.0, 4 mM DTT, 3 mM dithionite,
30 mM KCl, 2 mM AdoMet and 10,000-50,000 cpm of the spore photoproduct DNA
from pUC 18. This assay mix was placed in o-ring sealed eppendorfs and allowed to
incubate at 37 °C for 1 hour. After the incubation, 0.5 mL trifluoroacetic acid was added
to each assay mixture and transferred to quartz ampoules. These ampoules were sealed
under vacuum and placed in the oven at 165 0C for 3 hours. Then the seals of ampoules
were broken and trifluoroacetic acid was evaporated under the Schlenk line. Water (100
uL) was used to resuspend the solid. The resulting suspension was centrifuged at 12,000
rpm for 4 minutes and transferred to new eppendorf tubes. HPLC was used to separate
hydrolyzed DNA and protein by loading 15 uL of the assay onto a Waters phenyl-
Spherisorb column (4 mm x 25 mm) and running with water as mobile phase at 1.5

mL/min for 25 minutes. Fractions were collected every minute.

111.2.4 Re-irradiation of thymine photoproducts

For the re-irradiation experiments previously irradiated and hydrolyzed 3H-
labeled DNA was placed on SaranWrap and irradiated at 254 nm (UV GL—25, 2x4 W,
UVP) for 2 or 4 hours. The following product was loaded onto a Waters phenyl-
Spherisorb column (4 mm x 25 mm) running with water as mobile phase at 1.5 mL/min
flow rate for 25 minutes. Elution was monitored by absorption at 254 nm. Fractions were

collected every minute.

28

111.2.5 Scintillation Counting
Safety-Solve (15 mL) was added to each fiaction from HPLC chromatographic
runs. Then all the fractions were analyzed by scintillation counting (Wallac 1414-001).

The first sample used as blank, contained Safety-Solve(15mL) and water (1 mL).

111.2.6 HPLC of DNA bases

The HPLC of DNA bases, adenine, guanine, thymine and cytosine, were carried
out as follows. DNA bases were dissolved in water and filtered through 20 um filter. The
solution (15 uL) was injected onto Waters-phenyl-Spherisorb column (4 mm x 25 mm)
and running water as mobile phase at 1.5 mL/min flow rate for 25 minutes. Elution was

monitored by absorption at 254 nm.

111.3 Results and discussion

There are several ways to generate spore photoproduct in DNA. SP can be
induced between two thymines while irradiating dry film DNA (10), DNA/ethanol
solutions (9), frozen aqueous DNA solutions (5, 43), and irradiating DNA/SASP mixture
(14). Experiments included various types of DNA, like calf thymus DNA (5), T7 DNA
(9), pUC19 DNA or Bacillus spores (12, 14), which were labeled with tritium and
irradiated with UV light. The following steps included the acid hydrolysis of DNA and
quantification of thymine dimers. The best results were obtained while irradiating dry
DNA films, where 5% of total thymine was converted into SP (44) and irradiating

DNA/SASP complexes, where 5.8% of total thymine was converted to SP (14).

29

For our experiment, we first measured the elution time of the DNA bases from
HPLC chromatography. The standard samples were injected onto the Waters-phenyl-
Spherisorb column (4 mm x 25 mm) and elution was monitored by absorption at 254 nm.
Adenine, cytosine and thymine eluted at 2.5 minutes and guanine eluted at 7.5 minutes
(at a flow rate of 1.5 mL/min) (Table 111.1). DNA that we chose for our experiments was
pUC18 DNA due to its Similarity to pUC19 DNA used to generate SP by Nicholson and
co-workers (14). In order to make spore photoproduct, several experiments were carried
out. The first one involved irradiating dry film DNA at 254 nm, the second involved
irradiating 80% ethanol/DNA solution, and the third involved irradiating a DNA/ethanol
dry film. The DNA was irradiated for 5 hours at 254 nm, with the total UV dosage of 130
kJ/mz. When dry film DNA was irradiated, 3.8% of total thymine was converted to TT
dimer (based on data from the scintillation counting). When DNA/ethanol dry film was
exposed to UV light, 2.9% of total thymine was converted to TT dimer. However, when
80% ethanol solution of DNA was irradiated for 2 hours, only 0.3% of total thymine was
converted to TT dimer (Figure 111.1). The Similar results were obtained when 80%
ethanol solution of DNA was irradiated at 254 nm for 5 hours (data not Shown).
However, these results did not indicate which of the thymine dimers is formed -

cyclobutane dimer, (6-4) photoproduct or SP.

30

 

 

 

 

 

DNA base Concentration, mgmL Ehrtion time, min
NH2
N
</ I j Adenine 0.036mg/mL 2.5
3 ~/
NH;
\
I /L Cytosine 0.036 mg/mL 2.5
N O
H
O
N
</ I NH Guanine 0.07 mg/mL 7.5
N N/ NH2
0
l j; Thymine 0.026mgmL 2.5
N O
H

 

 

 

 

 

Table 111.1 DNA bases and their elution times from HPLC (Waters phenyl-Spherisorb

column). Eluent — water, flow rate — 1.5 mL/min, injection volume — 15 uL.

31

 

T'I‘ Dimer Production Under Difl’erent
Conditions

 

 

20000 — Dry Film DNA - 5 hr I

i
I — 80% EtOH/DNA - 2 hr
1

— DNA/EtOH Dry Film- 5 hr J
15000 *

 

 

 

5000- - - - -

 

 

 

 

 

Fractions

 

 

Figure 111.1 Thymine dimer production depending on conditions. Waters phenyl-
Spherisorb column (4 mm x 25 mm) was running with water as mobile phase at 1.5
mL/min flow rate for 25 minutes. Fractions were collected every minute. Thymine eluted

after 3—4 minutes and thymine photoproducts eluted after 8-9 minutes.

In order to investigate how UV dosage affects formation of thymine dimers the
following experiment was carried out. Based on research done by Nicholson and co-
workers (44), our next experiments involved irradiation of dry 3H-labeled DNA films at
254 nm for Shorter time — 10, 20, and 30 minutes (UV dosage 4, 8 and 12 kJ/mz,
respectively). However these yielded no thymine-thymine photoproducts. The difference
between the experiment carried by Nicholson and co-workers and ours was that they used
DNA/SASP mixture and that could be a major reason that there was no SP formed in our

experiment (Data not Shown).

32

Since the irradiation of DNA dry film gave the highest yield of TT photoproduct,
the next experiment was done by using photoproducts from dry film DNA. For the repair
assay, 50 uL of DNA (9.3 pmol) were mixed with 2 pl (238 pmol) of SPL and other
components as described in Materials and Methods, and incubated at 37 °C for different
amounts of time. Then the samples were quenched with TFA and acid hydrolysis was
performed. After hydrolysis the TFA was evaporated and the samples were resuspended
in 100 uL of water. Each sample (15 uL) was injected onto a Waters phenyl-Spherisorb
column and fractions were analyzed by scintillation counting. From the results (Figure
111.2) we can see that TT photoproduct not treated with SPL elutes at 13-14 minutes; if
incubated with SPL the peak elutes at 10-11 minutes. No significant repair was observed
and therefore it might be possible, that TT dimer was not Spore photoproduct. The
difference in elution time between DNA and DNA/SPL mixtures could be explained the
conformational change of thymine dimer or some unknown reasons. Other reason for the
doubt was that under the same irradiation conditions thymine photoproduct was not
reproducible, i.e. it eluted at different times from HPLC chromatography (the data not
shown).

In order to investigate the hypothesis that the photoproduct eluting at 13-14
minutes was not SP, the following experiments were done. DNA, which was previously
irradiated and hydrolyzed, was re-irradiated at 254 nm for 2 and 4 hours. Previous papers
Show (14, 45), that if cyclobutane dimers or (6-4) photoproducts are irradiated after acid
hydrolysis, they are converted to thymines, while spore photoproduct remains unchanged.

The results from re-irradiation, either after 2 hours or 4 hours indicate that there

were some changes between thymine-thymine dimers (Figures 111.3 and 111.4). The

33

unidentified thymine dimer eluted at 8-9 minutes before re-irradiation, but after 2 hours
of re-irradiation it eluted at 11-12 minutes (Figure 111.3). If the same DNA was re-
irradiated for 4 hours, thymine dimers eluted after 9-10 minutes. This indicates that
thymine dimer underwent some conformational changes or the thymine dimer was
converted into new thymine dimer and therefore, it might be Spore photoproduct.
However, these results need further investigations, like mass Spectrometry of thymine

dimers.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

T
SP Repir Assay
5000 I ------- DNA w/o SPL
I DNA w/ SPL after 20 rrin
I DNA w/ SL after40min
4000- , A A I.
I DNA w/ SPL after 60 min
i, DNA w/ SPL after 80 min
1
3000 - A AA A A-
2'
a.
U :
2000- 7 f i - I
1000 — 7
l
I . __. ,
O 20 25
L Fractions

 

 

Figure 111.2 SP repair assay. 3H-labeled spore photoproduct was incubated with
SPL for 20, 40, 60, and 80 minutes at 37 oC. Waters phenyl-Spherisorb column (4 mm x

25 mm) was running with water as mobile phase at 1.5 mL/min flow rate for 25 minutes.

34

Fractions were collected every minute. Thymine eluted after 3—4 minutes and thymine

photoproducts eluted after 8-9 and 12 minutes.

 

TT Photoproducts Before and After 2 hr 7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

I
I Reirradiation
l lOOOO
I j Sanple l - before
I
i 'I ' ‘ Sarrple 2 - before
8000 . A I 1 I
I I I -—--San'plel-after
I I T
l I ——--Sarrple2-afier
6000 . A ' A --
E
. a. i
l U I
I 4000 I
I I
l
I 2000 r l -
l
l
I j
I
0 . 4 , m
j 0 5 10 15 20 25
‘ Fractions

 

 

Figure 111.3 Thymine dimers before and after 2 hours of re-irradiation at 254 nm
wavelength. Waters phenyl-Spherisorb column (4 mm x 25 mm) was running with water
as mobile phase at 1.5 mL/min flow rate for 25 minutes. Fractions were collected every

minute. Thymine eluted at 3-4 minutes, thymine dimers — at 8-9 and 11-12.

35

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TT Photoproducts Before and After 4 hr
Reirradiation
10000
I l I Sample 1 - Before
I I I
I I I Sanple Z-Befone
3000«~2 II 2 2 I
I I I ----Sanplel-Afier
I
I L ----Sarrple2-Afler
6000« :2 2-12-, , ,
z I
8 I
I
4000
2000
0 , , I‘_, Y,
I 0 5 10 15 20 25
I Fractions
L

 

 

Figure 111.4 Thymine dimers before and after 4 hours of re-irradiation at 254 nm
wavelength. Waters phenyl-Spherisorb column (4 mm x 25 mm) was running with water
as mobile phase at 1.5 mL/min flow rate for 25 minutes. Fractions were collected every

minute.

36

CHAPTER IV

SPORE PHOTOPRODUCT LYASE BINDING T0 DNA AND
OLIGONUCLEOTIDES

IV. 1. Introduction

The enzyme SP lyase is the first identified non-photoactivatable pyrimidine dimer
lyase, and, together with the SASP’s, confers on Bacillus spores their unusual resistance
to UV radiation (18). SP lyase shares some sequence homology with the members of the
DNA photolyase/(6-4) photolyase/blue light photoreceptor protein family (18). The
conserved residues are in the general region believed to be involved in substrate binding
by DNA photolyase (31), although no structures of DNA photolyase bound to pyrimidine
dimers have been published. Computer-assisted comparisons performed between the
deduced amino acid sequence of SP lyase and other DNA repair proteins revealed that a
subset of the carboxyl half of SP lyase (amino acids from 170 to 250) shares sequence
homology with the DNA photolyases from number of prokaryotes and lower eukaryots,
suggesting that these two classes of enzymes may have descended from common
ancestral protein (46).

The involvement of DNA repair systems in spore resistance has been studied most
thoroughly for 254 nm radiation, where spores lacking NER or SP-specific repair are 2-3
times more UV-sensitive than wild-type spores (11). The NER system in B. subtilis
appears generally similar to that in E. coli and the gene products involved are also similar
(33). The NER system involves the products of a number of different genetic loci, while

SP—specific repair is due only to products of the spl locus. Cloning and mapping of DNA

37

that complements an SP lyase-defective mutant revealed that the SP lyase (spl) locus is

located at 118° on the B.subtilis map, immediately downstream from ptsI-II operon, which

 

 

SPL SPLDKRIEAAVKVAKAGYPLGFIVAPIYIHEGWEEGYRHLF 250

 

N . C . WSYNVDHFHAWTQGRTGFPI IDAAMRQVLSTGYMHNRLRMI 4 7 7
S . C . WENNPVAFEKWCTGNTGIPIVDAIMRKLLYTGYINNRSRMI 4 5 4
E . C . WQSNPAHLQAWQEGKTGY P IVDAAMRQLNSTGWMHNRLRMI 3 4 6
H . h . WRDDPAALQAWKDGETGYPIVDAGMRQLRAEAYMHNRVRMI 3 5 3
A . n . WENREALFTAWTQAQTGY P IVDAAMRQLTETGWMHNRCRMI 3 5 3

S . g . WRSDADEMHAWKSGLTGYPLVDAAMRQLAHEGWMHNRARML 3 3 4

 

Figure IV.1. One of the regions of sequence homology between SPL and DNA
photolyases from Neurospora crassa (N.c.), Saccharomyces cerevisiae (S. c. ), Escherichia
coli (E.c.), Halobacterium halobium (H.h.), Anacystis nidulans (A.n.), Streptomyces
griseus (S.g.). The conserved amino acids are shown in bold. The potential SPL binding
site to DNA (helix-tum-helix) is underlined (47).

is the part of phosphoenolpyruvatezsugar phosphotransferase (PTS) system (48).
Nucleotide sequence of the B.subtilis spl locus revealed that it is a two-gene operon,
where the first gene (splA) encodes a protein of nearly 9 kDa and which in some way
regulates expression of spl operon, and the second gene, splB, encodes an SP lyase, a
protein of nearly 40 kDa (18, 49). The spl operon is expressed only in sporulation in the
developing forespore, and is expressed in parallel with a/B-type SASP and transcribed by
RNA polymerase with the same forespore—specific sigma factor (06) that transcribes ssp

genes (18). The spl operon is not expressed during spore germination and is not induced

38

 

by DNA damage in growing cells (48). The analysis of splAB operon revealed that (a) SP
lyase may function in the cell as (at least) a homodimer, and (b) iron appears to be
necessary for optimal SP lyase activity during spore germination (48, 50). Also, spores of
mutants lacking the entire splAB operon exhibited the same UV sensitivity as spores
lacking only splB and only deletion of splB resulted in loss of SP lyase repair capability
(48).

It was shown that SplB containing a C-terminal lOHis tag purified from an E.coli
overexpression system bound to 35—bp double stranded DNA oligonucleotide containing
a single SP. However, this (lOHis)Sp1B was unable to complete catalysis unless its N-
terminal lO-His tag was first proteolytically removed at the engineered Xa site (44).
Thus, the SP-specific binding and cleavage functions of SP lyase appear to be separable.
SPL also specifically cleaved SP, but not cis,syn cyclobutane dimer, probably due to the
fact that (10His)SplB does not bind to pyrimidine dimer-containing DNA with high
affinity(44).

Although the three dimensional structure of DNA containing SP has not yet been
elucidated, it is known that cis,syn cyclobutane dimer distorts DNA, producing a helical
kink of 27° and unwinding of 19.70 (2). Because SPL binds SP but not Pyr<>Pyr with
high affinity, it presumably recognizes an SP-specific helical distortion in DNA which
differs in its geometry from distortion caused by cis,syn cyclobutane dimer (44). Binding
of SPL to SP apparently introduces additional distortion in the helix (44). Enhancement
in distortion of Pyr<>Pyr containing DNA by binding of DNA repair proteins has also
been seen in the Uvr(A)BC exonuclease, DNA photolyase, and phage T4 endonuclease

V. Once SP-specific binding occurs, the [4Fe-4S] cluster of SP lyase interacts with

39

AdoMet, presumably resulting in the creation of a 5’-adenosy1 radical, which participates
in reversal of SP to two thymines, likely by radical fragmentation (44).

Despite what we know about DNA photolyase and how it recognizes and binds to
DNA, there is very little knowledge about how SPL binds to DNA. Therefore a lot of
research has to be done to investigate SPL-DNA interactions, how SPL recognizes DNA

damage and if SPL binding depends on DNA sequence.

IV .2. Experimental methods
IV.2.1. Materials

Restriction enzyme EcoR I was purchased from New England BioLabs. The
oligonucleotides J C1, J C2, TK4-72a, and TK4-72b were purchased from Integrated DNA

Technologies. [y-32P]-ATP (10 uCi/ILL, 3000 Ci/mmol) was purchased from MP

Biomedicals.

IV.2.2 Agarose gel-based gel-shift assay
IV .2.2.1 Digestion of pUC18 DNA

NE buffer (9 uL) (50 mM NaCl, 100 mM Tris-HCl, 10 mM EDTA, 1 mM
dithiothreitol, 0.15% Triton X-100, 200 ug/ml BSA and 50% glycerol), 9 uL BSA, 6 ILL
EcoR I (111 units), 66 uL pUC18 DNA (40 pg) (total volume 90 uL) were mixed and
incubated at 37 °C for 1 hour. Then the reaction mixture was inactivated at 65 °C for 20

minutes. The linear DNA was stored at —20 °C.

IV.2.2.2 The extraction of linear pUC 18 DNA fi'om agarose gel

40

Linearized pUC18 DNA was purified from a 1% agarose gel (lg of agarose, 100
mL TAE Buffer and 30 uL ethidium bromide (10 mg/mL)) as follows. Loading dye (2
uL) was added to 25 uL of DNA mixture and loaded onto agarose gel. The standard 1 kb
DNA Ladder (500 pig/mL, New England BioLabs) was also loaded. A 100 V current was

applied to the gel. DNA was extracted using the Compass DNA Purification Kit

(American BioAnalytical) and stored at —20 °C.

IV 2.23 SPL binding to pUC 18 DNA

An agarose gel (0.5 %) was made of 0.5 g of agarose, lOOmL of TAE Buffer and
30 ILL ethidium bromide (10 mg/mL). The samples, containing either circular or linear
pUC18 DNA, were mixed in the Coy anaerobic chamber at 4 °C. The samples also
contained 4 uL of 50% glycerol and 2 uL of binding buffer (10 mM Tris-HCl pH 7.5, 50
mM NaCl, 0.5 mM DTT). Then samples were incubated for 2 hours at 4 °C. Then 2 uL

of loading dye was added to each of the sample and mixed. The samples were

electropheresed through 0.5% agarose gel at 100 V and 4 °C.

IV.2.3 Gel-shift assay using polyacrylarnide gel electrophoresis
IV 2.3.] The 5’-end labeling of 35-bp oligonucleotide

Two complementary oligonucleotides were synthesized (Integrated DNA
Technologies), one 35-mer containing a single pair of adjacent thymines (JCl
oligonucleotide, MW = 10,749 Da, 5’- CCC GGG GAT CCT CTA GAG TTG ACC

TGC AGG CAT GC —3’), and its complementary oligonucleotide (JC2 oligonucleotide,

41

MW = 10,758 Da, 5’- GCA TGC CTG CAG GTC AAC TCT AGA GGA TCC CCG GG
—3’)).

JCl oligomer (1 uL, 5.8 pmoles or 0.17 pg) was added to 5X Exchange Reaction
Buffer (Invitrogen), 5 IIL [II-321)] ATP (10 pCi/uL, 3000 Ci/mmol), 0.5 ILL T4
polynucleotide kinase (Invitrogen), 13.5 ILL autoclaved MQ water. Total volume was 25
uL. The mixture was incubated for 10 minutes at 37 °C followed by heat inactivation at
65 °C for 10 min. Ethanol precipitation was carried out as follows. To the Eppendorf tube
containing labeled oligomer was added 250 uL 4.67 M NH40Ac and 750 ILL 100 %
ethanol (ice cold). Then the sample was kept on ice for 30 minutes and centrifuged at
12,000 rpm for 20 minutes. The supernatant was removed and 500 ILL of 80 % ethanol
(ice cold) was added. The sample was centrifuged at 12,000 rpm for 20 minutes and
supernatant was removed. The oligomer was then dried under a N2 stream and
resuspended in 50 uL TAE Buffer. The labeled strand was hybridized to the unlabeled
complementary strand by heating at 90 0C for 2-3 minutes and then allowing to cool
down to room temperature. The hybridized oligo was purified using a mini Quick Spin

Oligo Column (Roche Applied Science). The oligo was stored at —80 °C in the freezer.

IV .232 Gel-shift assay of 35-bp oligonucleotide

The gel-shift assay reactions were prepared in a total volume f 20 ILL containing
50% glycerol, 10 mM Tris-HCl pH 7.5; 0 or 50 or 200 mM NaCl, 0 or 0.5 or 2 mM DTT,
0.1% NP-40, poly(dI-dC) (to prevent non-specific interactions — omitted for some
experiments), 0.22 pmol of the labeled double-stranded oligonucleotide, and spore

photoproduct lyase. The assay reactions were prepared anaerobically in an Mbraun glove

42

box and incubated for 1 hour at room temperature. The assays were electrophoresed
through a 12% or 8% non-denaturing polyacrylarnide gel at 20 V/cm. Then the gel was
taken out and placed in a dish with 7% acetic acid for 10 minutes. After that the gel was
placed between two gel drying films, put in the frame and dried overnight. The
electrophoresis products were visualized by autoradiography (X-Omat AR-5 film

(Kodak)).

IV.2.3.3 5’-End labeling of 94-bp oligonucleotide

Two other complementary oligonucleotides were synthesized (Integrated DNA
Technologies), based on Bacillus subtilis sequence (from 322456 to 322550).
Oligonucleotides had 94 nucleobases:

TK4—72a (MW = 28,902 Da) 5’- CGG GAT CAA CCA GAG CAT CAT GCT TGC GTT
ATC AAT GGT TGT TAT CGC CGC AAT GGT CGG TGC ACC GGG ACT TGG
TTC TGA AGT ATA CAG TGC C —3’ and
TK4-72b (MW = 29,057 Da) 5’- GGC ACT GTA TAC TTC AGA ACC AAG TCC
CGG TGC ACC GAC CAT TGC GGC GAT AAC AAC CAT TGA TAA CGC AAG
CAT GAT GCT CTG GTT GAT CCC G -3’

TK4-72a oligomer (1 uL, 5.8 pmoles or 0.17 ug) was added to 5X Exchange
Reaction Buffer (Invitrogen), 5 “L [II-3213] ATP (10 poi/ILL, 3000 Ci/mmol), 0.5 IIL T4
polynucleotide kinase (Invitrogen), 13.5 ILL autoclaved MQ water. Total volume was 25
ILL. The mixture was incubated for 10 minutes at 37 °C followed by heat inactivation at
65 °C for 10 min. Then oligomer was purified by ethanol precipitation as described

above.

43

IV.2.3.4. Gel-Shift assay of 94-bp oligomer

The 8% polyacrylamide gel was prepared using 10 mL 40% acrylarnide stock
solution, 1 mL 50X TAE Buffer, 0.35 mL ammonium persulfate (0.1 g/mL) and 17.5 uL
TEMED. The mixture was poured between two glass plates using pipette. The gel
polymerized within 1 hour. The gel then was pre-electrophoresed (100 V, 10 V/cm).

The samples were mixed in the Coy anaerobic chamber at 4 °C and were
incubated for 2 hours at 4 °C. Then 2 ILL of loading dye were added to each sample and
the samples were loaded onto the gel. Gel was run for 3 hours (200 V, 20 V/cm). Then
the gel was taken out and placed in a dish with 7% acetic acid for 10 minutes. Afier that
the gel was placed between two gel drying films, put in the frame and dried overnight.
The electrophoresis products were visualized by autoradiography and shown in figures

IV.5 and 1V.6.

IV.3 Results and discussion

The gel-shifi assay provides a method for detecting DNA-binding proteins. The
assay is based on the observation that complexes of protein and DNA migrate through a
gel more slowly than free DNA fragments or double-stranded oligonucleotides. The gel-
shifi assay is performed by incubating a purified protein with a non-labeled DNA or 32P
end-labeled DNA fragment containing the putative protein-binding site. The reaction
products are then analyzed on an agarose or on a non-denaturing polyacrylamide gel. The
specificity of the DNA-binding protein for the putative binding site is established by

competition experiments using DNA fragments or oligonucleotides containing a binding

44

site for the protein of interest or other unrelated DNA sequences. SPL binding properties
were tested using pUC18 DNA and short oligonucleotides. Two types of gels were used
for this experiment — agarose and polyacrylamide. Agarose gel was chosen because of the
experiment’s simplicity and because DNA doesn’t require radioactive labeling.
Disadvantage of this method is that it is not as accurate as polyacrylarnide gel, but it is
sufficient for the preliminary studies. The resolving power of polyacrylarnide gel is much
greater and it can separate molecules of DNA whose length differ by as little as 0.2%
(ref). For polyacrylarnide gel-shift assay two types of short oligonucleotides were used.
The 35-bp length oligonucleotide had only two adjacent thymines; the 94-bp length
oligonucleotide had more than two adjacent thymines. The experiments were carried to
investigate if SPL binding to DNA, if this binding is specific and if it depends on the

number of adjacent thymines.

IV.3.1 Agarose gel-based gel-shifi assay

First SPL binding properties were tested on circular pUC18 DNA. However, the
results were somewhat not reliable due to DNA tendency to be in two forms —
supercoiled and nicked which caused appearance of two bands on agarose gel (data not
shown). Therefore for the next experiment we included only supercoiled DNA purified
separately from the nicked DNA. The other type of DNA used for this experiment was
linear pUC18 DNA. The samples, containing pUC18 DNA and different amounts of
binding buffer (Table IV.1), were mixed with SPL and incubated for l or 2 hours and
loaded onto agarose gel. Lanes 1-7 had supercoiled pUC18 DNA, lane 8 had a 1 kb DNA

Ladder standard, and lanes 9-12 contained linear pUC18 DNA. For the supercoiled DNA

45

the effect of salt concentration (binding buffer) was also tested. Therefore lanes 1-4 had 2
ILL of binding buffer and lanes 5-7 contained no binding buffer (Figure IV.2). Lanes 1, 5
and 9 had no SPL; lanes 2-4, 6-7 and 10-12 had increasing number of protein. Band
shifiing in lanes 2-4, 6-7, and 10-12 clearly indicates SPL binding; DNA bands were
more retarded as the concentration of enzyme was increased. For supercoiled DNA,
however, we can see two bands (lanes 1 and 5), which suggest that some of the DNA
became nicked even though supercoiled DNA was purified from nicked DNA prior
running the gel. Therefore it is hard to say if SPL is binding supercoiled DNA, nicked
DNA or both (from the results we can see, that both' bands were shified afier adding SPL
- lanes 2-4 and 6-7). The other problem that occurred, was that some of the DNA, both
supercoiled and linear, were stuck on the wells of the gel. The reason of that could be the
aggregation of SPL and to avoid this problem for later experiments the DNA and protein
mixtures were incubated at 4 °C temperature and the gel was run at 4 °C as well. Due to
instability of supercoiled DNA, later experiments included only linear pUC18 DNA,
which was cut with the restriction enzyme EcoRI and purified from agarose gel.

For the second gel (Table IV.2, Figure IV .3) lane 1 had no SPL and lanes 2-5 had
increasing amount of enzyme; lane 6 had a 1 kb DNA Ladder standard. The second gel
also had broader concentration range than the previous one. From this gel we can see that
much less of DNA-protein adduct is stuck on the wells of the gel. Also, one SPL
molecule can bind up to 12 DNA base pairs, which means that SPL might be binding not
only to thymines. From these results we can make several conclusions, (i) SPL does bind

to DNA, (ii) SPL binding might be non-specific, (iii) SPL can dimerize or bind to itself.

46

Table IV.1 DNA and SPL binding conditions for agarose gel in Figure IV.2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lanes
Supercoiled DNA Std. Linear DNA

1 2 3 4 5 6 7 8 9 10 11 12
DNA, [AL 3 3 3 3 3 3 3 3 3 3 3
Binding Buffer, “L 2 2 2 2 0 0 0 2 2 2 2
50% Glycerol, “L 4 4 4 4 0 0 0 4 4 4 4
MQ Water, pL 11 9 7 5 17 15 13 ll 9 7 5
SPL. IIL 0 2 4 6 0 2 4 0 2 4 6
Cone. of SPL, nrrol 0 0.04 0.09 0.14 O 0.04 0.09 0 0.04 0.09 0.14
Molecules per base pair 0 0.027 0.055 0.083 0 0.027 0.055 0 0.02 0.04 0.077

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

123456789101112

 

 

 

 

Figure IV.2 Agarose gel of circular pUC18 DNA (lanes 1-4), supercoiled pUC18 DNA
(lanes 5-7) and linear pUC18 DNA (lanes 9-12), standard — lane 8.

47

Table IV.2 Linear DNA and SPL binding conditions for agarose gel in Figure IV.3

2 4 6 8
Cone. mm] 0.04 0.09 0.14 0.18
Mobcules base 0.02 0.04 0.077 0.083

 

 

 

Figure IV.3 Agarose gel of linear pUC18 DNA (lanes 1-5); standard — lane 6. The

samples were incubated and the gel was run at 4 0C.

48

IV.4.2 Gel-shift assay using polyacrylamide gel electrophoresis

The gel-shift assays were done in order to confirm, that SPL binds to DNA and to
find the binding constant. Binding assays were carried out for two different
oligonucleotides, 35-bp with only two adjacent thymines and 94-bp with several adjacent
thymine dimers. Also, the SPL binding was tested for non-irradiated oligomers and
irradiated at 254 nm oligomers.

When 35-bp oligomer was not purified, lanes 1-3 and 7-9 (Table IV.3, Figure
IV .4), it had too much contamination, while the oligomer, which was purified with mini
Quick Spin Oligo Column (lanes 4-6 and 10-12), had only oligomer and it’s adduct with
the protein. From this gel (Figure IV.4) we can also see that while increasing the amount
of protein the binding increases as well.

The next gel (Table IV .4 and Figure IV.5) contained only purified oligomers. Half
of the samples had oligomers, which were non-irradiated (lanes 1-6) and another half
with irradiated at 254 nm oligomers (lanes 7-12). Lanes 1 and 7 had only oligomer and
no SPL, while lanes 2-6 and 8-12 had increasing amount of enzyme. From the results we
can see, that SPL equally binds both irradiated and non-irradiated oligomers; binding
increases while increasing the concentration of protein. The only problem was, that all
bound oligomer stayed on the wells without going into the gel.

The next experiment included a 94-bp oligomer with more adjacent thymines and
lower percentage acrylamide gel (8% instead of previous 12%). The other conditions that
were changed, was the concentration of salt (low salt and high salt or even no salt), as
well as skipping the poly(dI-dC) for some of the samples (Table IV.5 and Figure 1V.6).

The incubation was carried out at 4 °C for 1 hour and gel was running at 4 °C to help to

49

prevent the precipitation or aggregation. The best binding was achieved, when reaction
mix had low concentration of salt and no poly(dI-dC), which probably binds to SPL as
well.

The gel-shift assay was repeated with oligomer at low concentration of salt and
decreased concentration of poly(dI-dC) (10 fold, lanes 1 to 7). Lanes 8-14 had no
poly(dI-dC). Lanes 1 and 8 had no enzyme; lanes 2-7 and 9-14 had increasing amounts of
the SPL (Table IV .6 and Figure IV.7). It was observed, that the binding is better without
the poly(dI-dC) and the full binding was observed when the concentration of SPL was 0.8
molecules of enzyme per base pair of oligomer.

The conclusions from this set of the experiments are, (i) the change of acrylamide
concentration from 12% to 8% did not change the results and DNA-SPL adducts were
stuck on the wells of the gel, (ii) the temperature change for the incubation (fi'om 22 to 4
°C) also did not make big difference, (iii) the best binding was observed at low salt

concentration.

50

Table IV.3 The binding conditions for gel-shift assay

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lanes
Non-irradiated Oligo Irradiated Oligo
Non-purified Purified Non-purified Purified
1 2 3 4 5 6 7 8 9 10 ll 12
Binding Buflier, IL 2 2 2 2 2 2 2 2 2 2 2 2
50% Glycerol, uL 4 4 4 4 4 4 4 4 4 4 4 4
poly (dI-dC), “L 1 l 1 1 l 1 l 1 1 1 1 1
DNA, “L 2 2 2 2 2 2 2 2 2 2 2 2
MQ Water, “L 11 10.8 10.6 10.4 10.2 10 11 10.8 10.6 10.4 10.2 10
SPL, “L 0 0.2 0.4 0.6 0.8 1 0 0.2 0.4 0.6 0.8 1
Cone. ofSPL, nmol 0 2.4 4.8 7.1 9.5 11.9 0 2.4 4.8 7.1 9.5 11.9
Molecules per oligo 0 109 218 318 437 583 0 109 218 318 437 583

 

 

 

 

 

 

 

 

 

 

Figure IV.4 Gel-shifi assay of the 32P-labeled 35-bp oligonucleotide. Lanes 1 to 6 had
non-irradiated oligomer and lanes 7 to 12 had irradiated oligomers. Samples 1-3 and 7-8
were not purified and the samples 4-6 and 10-12 were purified by using mini Quick Spin

Oligo Column (Roche Applied Science).

51

Table IV.4 The binding conditions for gel-shift assay

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Lanes
Non-irradiated Oligo Irradiated OligrLSP—
1 3 3 4 5 6 7 8 9 10
Binding Buffer, “L 2 2 2 2 2 2 2 2 2
50% Glycerol, “L 4 4 4 4 4 4 4 4 4 4
poly(dI-dC), “L 1 1 1 1 1 1 1 1 1 1
1% NP-40, “L 1 1 1 1 1 1 1 1 1 1
Oligo, ”L 1 1 1 1 1 1 l 1 1 1
MQ Water, “L 11 10.6 10.4 10.2 10 11 10.6 10.4 10.2 10
SPL, “L 0 0.4 0.6 0.8 1 0 0.4 0.6 0.8 1
Cone. ofSPL, ml 0 4.8 7.1 9.5 11.9 0 4.8 7.1 9.5 11.9
Molecules per oligo 0 437 700 875 1166 0 437 700 875 1166

 

 

Figure IV.5 Gel-shift assay of the 32P-labeled 35-bp oligonucleotide. Lanes 1-5 had non-
irradiated oligomer and lanes 6-10 had irradiated oligomers. Lanes 1 and 6 had no SPL;

lanes 2-5 and 7-10 had increasing amounts of SPL.

52

Table IV.5 The binding conditions for gel-shifi assay

With
Low Salt
3

y—i
O

5 3 l
2.26 6.78 11.3
19 58 95

ll 10 8 6 4 14 13 ll 9

Conc. 0 2.26 6.78 11.3 15.8 0 2.26 6.78 11.3
Molecubs 0 19 58 95 134 O 19 58 95

OOQOHt—‘O-boo

 

Figure IV.6 Gel-shift assay of the 32P-labelcd 94-bp oligonucleotide. Lanes 1-5 are the
samples incubated with poly (dI-dC) at low ionic strength (10 mM Tris-HCl pH 7.5, 50
mM NaCl, 0.5 mM DTT); lanes 6—9 are the samples that had no poly(dI—dC) and no salt;
lanes 10-13 are the samples incubated without poly(dI—dC) at high ionic strength (40 mM
Tris-HCl pH 7.5, 200 mM NaCl, 2 mM DTT). Lanes 1,6,10 had no SPL.

53

13

12 11
0 l 3 4
3 0 2.26 4.52 6.78 9.04 1

ll 10
0 1 2 3 4
0 2.26 4.52 6.78 9.04 l

7
2
4
l
l
l
5
6

6
2 2
4 4
1 0
1 1
l l
6 7
5 5
l. l.

3

 

Table IV.6 The binding conditions for gel-shifi assay

1 2 3 4 5 6 7 8

N ‘

 

Figure IV.7 Gel-shift assay of the 32P-labeled 94—bp oligonucleotide. Lanes 1-7 are the
samples that were incubated with poly (dI-dC) at low ionic strength (10 mM Tris-HCl pH
7.5, 50 mM NaCl, 0.5 mM DTT); lanes 8-14 are the samples that were incubated without
poly(dI-dC) at low ionic strength (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.5 mM DTT);
the lanes 1 and 8 had no SPL.

54

CHAPTER V

PHOTOSYNTHESIS OF SPORE PHOTOPRODUCT FROM
THYMIDINE

V.1 Introduction

DNA bases are the specific chromophores of nucleic acids in the ultraviolet
region of the spectrum. Unlike other chromophores involved in photobiology, such as
chlorophyll in photosynthesis or retinaldehyde in vision phenomena, some of the
photochemical reactions that are induced in nucleic acids by UV light give rise to
unwanted biological effects at the cellular level (3, 51). Those effects could be cell death,
photomutagenesis, and photocarcinogenesis. A bacterium lacking the normal repair
mechanism has a 0.5 death probability when only 10 molecular photochemical defects
are created per DNA molecule (~107 nucleotides)(6).

DNA structure contains four different bases — adenine (A), guanine (G), thymine
(T), and cytosine (C). Within nucleic acids, the hydrogen atoms linked to the nitrogen
N(l) of the purine bases (A and G) and to the nitrogen N(1) of the pyrimidine bases (T
and C) are substituted by a five-carbon sugar (2-deoxy-D-ribose in DNA). The complete
nucleic acid building blocks involve the presence of phosphoric acid. DNA damage is
induced by the direct absorption of incident UVB and UVC photons by nucleobases,
whose excited states can be populated by direct UV irradiation. As a result, dimeric
photoproducts, such as various isomeric cyclobutane type dimers, pyrimidine (6-4)
pyrimidone photoadducts, and 5,6-dihydro-5-(a-thyminyl)-thymine (spore photoproduct)

are produced between adjacent pyrimidine bases (Scheme V. l)(3).

55

HN

HO

   

0
\P// O
o/ \
H
H T<>T \\:nm 0
HN
| 0
HO 0 N
0 HN

I 312nm

 

HO

 

 

Spore photoproduct Dewar 'l'l‘

Scheme V.1 The formation of various thymidine photoproducts under UV irradiation

56

Cyclobutane pyrimidine dimers are generally the most common type of UV-
induced DNA damage in cells. Early experiments have shown, that the cyclobutane
pyrimidine dimers are no longer the main DNA lesions induced by monochromatic UV
light in highly dehydrated microorganisms such as bacterial spores(1 1), or when DNA is
irradiated either at low temperature or as a solid film (43, 52). Under these conditions the
major photoproduct is SP, which has two diastereomers (5R* and 58*) and which can be
separated on reversed phase or silica gel HPLC columns and characterized by FAB mass
spectrometry or 1H and '3 C NMR analysis (53). Interestingly, only one of the two
possible SP diastereomers was obtained when thymidine was irradiated as a dry film and
while both diastereomers were obtained in frozen solution (54). Different stereochemistry
of the photoreaction is due to different stacking of nucleosides in different forms of DNA
(5). Two mechanisms have been proposed for the formation of SP. The first one involves
the recombination of the photogenerated 5-(2’-deoxyuridyl)methyl and the 5,6-
dihydrothymid-S-yl radicals (53). From electron spin resonance studies many
investigators have reported the formation of thymyl radical (Scheme V.2, a (5)) in DNA
and thymidine as a result of ultraviolet irradiation. The thymyl radical is formed by the
addition of a hydrogen atom at the C(6) position of a thymine moiety. In situations such

as may exist in dry or frozen DNA or thymidine, the methyl group of an adjacent

o o o o 0
CH3 H2C
HN HN . ' NH HN NH
2% ' ”A “ ' )C’A ' A
O N O N H H N O o N N O
H H H H H
a b
Scheme V.2 The UV-light induced formation of thymyl and thyminyl radicals (55)

57

thymine residue may serve as the hydrogen donor. The resulting thyminyl radical (Figure
V.2, b) adds on to the thymyl radical, forming stable spore photoproduct (56).

The second proposed mechanism is a concerted mechanism (Scheme V3) (53),
which has been shown to take place in the frozen state while using methyl deuterated
[CD3] thymidine as a substrate (53, 54). Interestingly, the deuteration of the C(6) carbon
was found to take place in highly stereospecific way, since only one singlet
corresponding to one of to H(6) protons is observed in the 1H NMR spectrum of the
hexadeuterated diastereomers which has nonmagnetically equivalent methylene protons
(53). The photosensitization experiment of thymidine by benzophenone in the dry state
also suggests that a concerted mechanism is involved in the formation of SP in dry films
of thymidine (52, 54). Photosensitization is reaction when DNA damage can be resulted
from wavelengths in the electromagnetic spectrum, which, though not absorbed directly
by bases, are absorbed by other molecules (sensitizer molecules) that then transfer energy
to bases of in DNA. Sensitizing molecules can behave as a photocatalyst or it can be
consumed. The photosensitization reactions that damage DNA may include the formation
of thymine dimers by triplet excitation transfer. Various ketones promote pyrimidine
dimer formation, a notable example being acetophenone. The lowest triplet energy state
of acetophenone is slightly higher than that of thymine but lower that the triplet states of
other DNA bases (Scheme V.4). Upon irradiation of DNA in the presence of
acetophenone at wavelength of ~300 nm, the triplet energy state of the photosensitizer is
transferred to thymine, facilitating the formation of thymine dimers (1). However, some

investigators used benzophenone for the generation of the SP instead (54).

58

 

Scheme V.3 Concerted mechanism for the formation of the hexadeuterated 5,6-

dihydro 5-(0t-thymidyl) thymidine upon exposure to far-UV light in frozen aqueous
solutions (53).

 

I S1 T1
A
G,T -——-
C —
E
M '— (C;
c ..... _
T ....... i__ A
¢Ac T

 

 

 

 

Scheme V.4 The energy levels of the lowest excited singlet states (SI) and lowest
triplet states (T1) of adenine (A), guanine (G), cytosine (C), thymine (T), and
acetophenone (oAc) (Adapted from Ref. (1))

59

 

V.2. Experimental methods
V.2.l Materials

Thymidine and benzophenone were purchased from Si grna-Aldrich.

V.2.2 Recrystallization of thymidine and benzophenone

Benzophenone was recrystallized from ethanol. Benzophenone (100 mg) was
dissolved in minimum amount of ethanol while heating, filtered through filter paper
(Whatman) into an Erlenmayer flask, and the filtrate was placed on ice to crystallize. The
crystals were filtered using vacuum filtration, washed with a small amount of ice-cold
ethanol, and dried.

Thymidine (2 g) was added to 20 mL of ethanol and heated to boiling. Since the
solution was not clear, 10 mL of water and some activated carbon was added. This hot
mixture was filtered through paper filter into an ice-cold Erlenmayer flask kept on ice.

White precipitate formed. The precipitate was filtered and dried.

V.2.3 Photosynthesis of SP

The synthesis of spore photoproduct from thymidine in the solid state was carried
out as follows. Recrystallized thymidine (100 mg) and 50 mg of recrystallized
benzophenone were dissolved in 50 mL of ethanol in a 6” test tube (Pyrex). The ethanol
was then evaporated overnight in the fume hood. The samples were irradiated either in a
Rayonet photoreactor, which was equipped with 8 UV lamps (>290 nm) or in a custom

made photoreactor, which was equipped with 2x40 W 350 nm lamps (see Table V.l).

6O

Then some of the sample was dissolved in D20 and transferred to an NMR tube for 1H
NMR analysis.

The synthesis of spore photoproduct from thymidine in the liquid state was done
as follows. Thymidine (50 mg) and 25 mg of benzophenone were dissolved in 25 mL of
either ethanol or water and the argon was purged for 1 hour. Then the samples were
placed in photoreactor, which was equipped with 2x40 W 350 nm ultraviolet lamps and
irradiated for 21 hour. After the reaction was done, ethanol or water was evaporated
under the Schlenk line and some of the sample was dissolved in D20 for 1H NMR
analysis.

The third type of reaction involved irradiation of thymidine in frozen aqueous
solution. 100 mg of thymidine was dissolved in 16.6 mL of MQ water in a Petri dish. The
Petri dish with the solution was placed over dry ice and frozen. The solution was
irradiated with UVGL-25 UV lamp (4 W, 254 nm, UV Products) for 6 hours while
keeping the Petri dish over dry ice. After the irradiation was done, the sample was
thawed, the water was evaporated and some of the sample was dissolved in D20 for 1H

NMR analysis.
V.2.4 NMR spectroscopy

1H NMR spectra were recorded at room temperature on Varian Inova-300

spectrometer (300 MHz) or on Varian VRX-500 spectrometer (500 MHz).

61

V.3 Results and discussion

The UV-induced DNA damage usually is from the direct absorption of photons by
bases in DNA. However, photosensitizers, like acetophenone, benzophenone or other
ketones, can induce thymine dimers as well. For our experiments as a photosensitizer was
chosen benzophenone, based on work done by Douki and co-workers (54).

First, the 1H NMR of pure thymidine in D20 was carried out and it had following
peaks in 1H NMR spectra: 1.88 (3H, methyl), 2.32-2.38 (2H, H2’), 3.70-3.88 (2H, H5’),
3.98-4.40 (1H, H4’), 4.42-4.48 (1H, H3’), 4.84 (D20 peak), 6.24-6.30 (1H, Hl’), 7.64
(1H, H6). Because D20 was used a solvent, there are no peaks in spectrum representing
hydrogens from NH and 0H (Figure V.l).

The first set of experiments involved irradiation of thymidine/benzophenone in
solid state. Some of the reactions were carried out under argon to prevent the scavenging
effect of oxygen radicals. However, the irradiation of solid thymidine at >290 nm
resulted no photodimers (Table V. l ). Following experiment involved irradiation of frozen
aqueous thymidine solution at 254 nm, which also was unsuccessful. That could be due to
short irradiation time, weak UV source or poor interaction between thymidine and
benzophenone. The results are summarized in Table VI

Since the interaction between molecules is more intense, the next set of the
experiments included irradiation of thymidine/benzophenone ethanolic solutions. The
irradiation of thymidine/benzophenone in ethanol solution at 350 nm gave photoproduct,
which is likely to be (6-4) photoproduct. But the yield of the obtained photoproduct was
very low and from 1H NMR spectrum it was hard to confirm the identity of photoproduct

(see Figure V.l). Besides the peaks from the starting material thymidine, there were

62

additional peaks due to benzophenone 7 .20-7.50 and (6-4) photoproduct - 2.32-2.38 (1H,
H2’), 2.65 (1H, H2”), 2.78 (3H, methyl), 2.94 (3H, methyl), 3.00 (1H, H2”), 3.10 (1H,
H2’), 3.80 (2H, H5’), 4.10 (1H, H4’), 4.42 (1H, H3’), 4.84 (D20 peak), 4.90 (1H, H6),
5.55 (1H, Hl’), 5.82, 5.88 (1H, Hl’).

But when thymidine/benzophenone ethanolic solution was irradiated for
prolonged time, 37 hours, some of the peaks, like at 2.78 ppm and 2.94 ppm, were gone,
which indicates of a loss of thymine dimer (Figure V.l). When the amount of
benzophenone (thyminezbenzophenone 1:1 ratio and 1:2 ratio) was increased to see if the
yield of unknown photoproduct can be improved, it gave no significant changes (spectra
not shown). The similar result to what was obtained fi'om irradiating thymidine at 350 nm
for 18 hours was obtained by irradiating thymidine in ethanol solution (no benzophenone
added) at 254 nm in a quartz test tube for 23 hours (Figure V.l). 1H NMR peaks of this
photoreaction are: 2.60 (1H, H2’), 2.65 (1H, H2”), 2.71 (3H, methyl), 2.89 (3H, methyl),
3.00 (1H, H2”), 3.10 (1H, H2’), 3.80 (2H, H5’), 4.10 (1H, H4’), 4.42 (1H, H3’), 4.84
(D20 peak), 4.90 (1H, H6), 5.50 (1H, Hl’), 5.85 (1H, Hl’). However, when the same
type of reaction was carried out in water, there was no photoproduct formed neither at

350 nm nor at 254 nm (Table V.l).

63

Table V.l Various conditions for making spore photoproduct from thymidine

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

" # Phase Argon Solvent Benzophenone 3. Time, h T<>T Dimer
1 Solid - EtOH + >290 nrn 22 -
2 Solid + EtOH + >290 nrn 24 -
3 Solid + EtOH + >290 nrn 24 -
4 Solid - EtOH + >336 nm 24 -
5 Frozen - Water - 254 run 6 -
6 Liquid - EtOH + >290 nrn 21 -
7 Solid - EtOH + 350 nm 24 -
8 Solid - EtOH + 350 nm 48 -
9 Liquid - Water - 350 nm 24 -

10 Liquid + EtOH + 350 nm 24 +
11 Liquid + Water + 350 nm 24 -
12 Liquid + Water - 254 nm 21 -
13 Liquid + EtOH - 254 nm 23 +
14 Liquid + EtOH 1:1 ratio 350 nm 18 +
15 Liquid + EtOH 1:1 ratio 350 nm 18 +
16 Liquid + EtOH 1:2 ratio 350 nm 37 +
17 Liquid + EtOH 1:2 ratio 350 run 37 +

 

64

 

| NH

 

 

 

3.10 3.00 2.902.130 2.70 I
ppm (t1)

 

 

 

 

 

 

 

 

 

 

3.103.002.90 2.802.70 I I
ppm «1) I I I 1
II I a. . .
‘4 L4 If” H I I0 LI!

 

3.10 3.00 2.90 2.80 2.70
ppm «0 I

 

 

 

 

PPM 01)

Figure V.1 1H NMR spectrum of thymidine and its dimers (in D20). Top spectrum —
thymidine in D20 (Varian Inova-300, 300 MHz), insert — the schematic representation of
thymidine; the second from the top — thymidine and benzophenone ethanol solution after
18 hours of irradiation at 350 nm (Varian VRX-500, 500 MHz); the third from the top —
thymidine and benzophenone ethanol solution after 23 hours of irradiation at 254 nm
(V arian Inova-300, 300 MHz); the bottom spectrum — thymidine and benzophenone
solution in ethanol after 37 hours of irradiation at 350 nm (Varian Inova-300, 300 MHz).

65

CHAPTER VI

CONCLUSIONS

Spore photoproduct lyase was overexpressed and purified under anaerobic
conditions as described; approximately 10 mg of purified protein was obtained from 6 L
of bacteria culture. This as-isolated SPL contains 1.86 mol F e/mol protein.

For SPL binding activity gel-shift assay experiments were established. The
preliminary binding studies were carried out between pUC18 DNA and SPL on an
agarose gel. It showed that SPL binds to DNA with the ratio 1 molecule of SPL to ~12
base pairs of DNA. The more insightul studies were done by using polyacrylarnide gel-
shift assays. Two types of double-stranded oligonucleotides were end labeled with 32P
and reactions were carried out. The first double-stranded oligonucleotide, which had 35
base pairs and had one adjacent TT pair, was found to bind to SPL. The second oligomer,
which had 94 base pairs, also was found to bind to SPL. Various assay conditions and
reactions were tried; the best conditions for binding were at low salt concentration and
without specific non-competitor poly(dI-dC). However, more work has to be done,
including quantification of bound oligonucleotides, finding SPL binding constant, and
determining how binding depends on the sequence of the DNA bases.

Attempts to generate spore photoproduct from tritium labeled DNA were also
made. It was found that best yield of thymine dimer was obtained under irradiation of dry
film DNA at 254 nm wavelength for 5 hours. However, further investigations failed to
prove that this photoproduct is spore photoproduct and more research has to be done,

including optimization of irradiation conditions to maximize photoproduct production. In

66

addition, analytical tools like NMR and mass spectrometry need to be used characterize
the photoproducts and correlate to specific peaks on the HPLC Chromatogram.

The effect of irradiation on thymidine was also carried out. The reactivity of
thymidine was tested under various conditions, including the experiments in solid state,
liquid phase and in frozen solution. The irradiation of thymidine/benzophenone ethanol
solution at 350 nm led to formation of a photodimer, which is likely to be (6-4)
photoproduct, but the yield of the reaction was very low and therefore it is hard to make a
conclusion. More research has to be done, including finding the conditions for creating
spore photoproduct. The ultimate goal of this work is to develop a more efficient method
of SP generation. The resulting SP could then be used for SP repair assays and other

experiments, like mass spectrometry etc.

67

10.

ll.

12.

13.

14.

15.

16.

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71

 

IIIIIIIIIIIIIIIIIIIII

 

 

— ._.-P.i‘—-_ ___. _ _