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This is to certify that the
thesis entitled
INHIBITION OF EUKARYOTIC DNA POLYMERASES BY
PHOSPHONOACETATE AND PHOSPHONOFORMATE

presented by

Carol Lee Kempen Sabourin

has been accepted towards fulfillment
of the requirements for

M.S. Biochemistry

degree in

 

 

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Major professor

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Date 10/27/77

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INHIBITION OF EUKARYOTIC DNA POLYMERASES BY

PHOSPHONOACETATE AND PHOSPHONOFORMATE

BY

Carol Lee Kempen Sabourin

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Biochemistry

1977

ABSTRACT

INHIBITION OF EUKARYOTIC DNA POLYMERASES BY
PHOSPHONOACETATE AND PHOSPHONOFORMATE

BY

Carol Lee Kempen Sabourin

Phosphonoacetate was found to be an inhibitor of the DNA polymerase
a from three human cells, HeLa, Wi-38, and phytohemagglutinin-stimulated
lymphocytes. The inhibition patterns were determined. The apparent
inhibition constants (Kii) were about 30 uM. Thus the DNA polymerase
a is 15 to 30 times less sensitive to phosphonoacetate than the
herpesvirus-induced DNA polymerase. The DNA polymerase a from Chinese
hamster ovary cells and calf thymus was also inhibited. The DNA
polymerases B and Y from the eukaryotic cells were relatively insensi-
tive to phosphonoacetate. The sensitivity of the DNA polymerase a and '
the relative insensitivity of the DNA polymerases B and Y appeared to
be a general characteristic of the vertebrate polymerases. DNA
polymerases from two other eukaryotic cells, yeast DNA polymerase A
and B and tobacco cell DNA polymerase, were inhibited by phosphono-
acetate. Fourteen phosphonate analogues were examined for inhibition
of the HeLa DNA polymerase a. Only one, phosphonoformate, was an
inhibitor. The mechanism of inhibition for phosphonoformate was

analogous to that for phosphonoacetate.

DEDICATION

To my parents and my husband, Pat

ii

ACKNOWLEDGEMENTS

The author wishes to express her deepest appreciation to Dr.
J. A. Boezi for his guidance, criticism, and encouragement which
made possible this study. Sincere appreciation also goes to the
members of my guidance committee, Dr. A. Revzin and Dr. L. F.
Velicer. I would also like to thank John Reno for his helpfulness
and Betty Baltzer for her technical assistance. Finally, I am
indebted to the Department of Biochemistry at Michigan State Uni-

versity and the National Cancer Institute for financial support.

iii

TABLE OF CONTENTS

Page
nwmmmnnm.. ... ... ... ... ... ... ... ... 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . 3

Vertebrate DNA Polymerases . . . . . . . . . . . . . . . 3
DNA Polymerase a. . . . . . . . . . . . . . . . . 3
DNA Polymerase B. . . . . . . . . . . . . . . . . 6
DNA Polymerase y. . . . . . . . . . . . . . . . . 8
DNA Polymerase Activities in vivo . . . . . . . . 10
DNA Polymerases During Development. . . . . . . . 12
Phosphonoacetate and Herpesvirus Infection . . . . . . . 14
Initial Studies with Phosphonoacetate . . . . . . 14
Phosphonoacetate Inhibition of Herpesviruses
and Other Viruses . . . . . . . . . . . . . . . l6
Phosphonoacetate Inhibition of the Herpesvirus-
Induced DNA Polymerase. . . . . . . . . . . . . 18
Effect of Phosphonoacetate on Other DNA
Polymerases . . . . . . . . . . . . . . . . . . 20
Effect of Phosphonoacetate on a Cell Line
Transformed by Epstein-Barr Virus . . . . . . . 21
Animal Studies with Phosphonoacetate. . . . . . . 24

EXPERIMENTAL PROCEDURES 0 .. o o o o o o o o o o o o o o o o o o 29

Reagents . .’. . . . . . . . . . . . . . . . . . . . . . 29
Cell Cultures. . . . . . . . . . . . . . . . . . . . . . 29
Assay of the DNA Polymerization Reaction . . . . . . . . 30
Purification of the DNA Polymerases. . . . . . . . . . . 31
Inhibition Patterns. . . . . . . . . . . . . . . . . . . 32

ESULTS O O O O O O O O O O O O O O O O I O O O O O O O I O O O 33

Phosphonoacetate Inhibition of the DNA Polymerization
Reaction Catalyzed by HeLa DNA Polymerase a. . . . . . 33
Phosphonoacetate Inhibition of the DNA Polymerization
Reaction Catalyzed by Wi-38 DNA Polymerase a . . . . . 38
The Effect of Phosphonoacetate on the DNA Polymeriza-
tion Reaction Catalyzed by Other a-Polymerases . . . . 38
The Effect of Phosphonoacetate on the DNA Polymeriza-
tion Reaction Catalyzed by Other DNA Polymerases . . . 41
The Effect of Other Phosphonate Compounds on the DNA
Polymerization Reaction Catalyzed by HeLa DNA
Polymerase a . . . . . . . . . . . . . . . . . . . . . 41

iv

DI SCUSS ION .

REFERENCES .

Figure

LIST OF FIGURES

Double reciprocal plots with the four dNTPs as the

variable substrate and phosphonoacetate as
of the purified HeLa a-polymerase. . . . .

Double reciprocal plots with activated DNA
variable substrate and phosphonoacetate as
of the purified HeLa a-polymerase. . . . .

Double reciprocal plots with activated DNA
variable substrate and phosphonoacetate as
of the purified Wi-38 a-polymerase . . . .

inhibitor

as the
inhibitor

as the
inhibitor

Double reciprocal plots with the four dNTPs as the

variable substrate and phosphonoformate as
of the purified HeLa a-polymerase. . . . .

inhibitor

Multiple inhibition of the HeLa a-polymerase by .

phosphonoacetate and phosphonoformate. . .

vi

Page

35

37

4O

44

46

LIST OF ABBREVIATIONS

HSV herpes simplex virus

EBV Epstein-Barr virus

IUdR 5-iodo-2'-deoxyuridine

dNTP deoxynucleoside triphosphate
Tris tris-(hydroxymethyl)-methylamine
DEAE . diethylaminoethyl

vii

INTRODUCTION

Phosphonoacetate is an effective inhibitor of the replication
of herpesviruses (l,2,3,4). The inhibition of viral replication is
through an effect on the viral—induced DNA polymerase (S,6,7,8,9).
At the concentrations at which phosphonoacetate is an effective anti-
herpesvirus agent, it has no obvious cytotoxic effects. At higher
concentrations, however, phosphonoacetate is cytotoxic (2,5,10) and
cellular DNA synthesis is inhibited. Cell growth is impaired and
the cells are apparently arrested at interphase.

Mao et a1. (6) and Mao and Robishaw (7) reported that the
inhibitory effect of phosphonoacetate on the herpesvirus-induced DNA
polymerase was specific. They reported that the herpesvirus-induced
DNA polymerase was highly sensitive to phosphonoacetate but that the
cellular DNA polymerases a and B of human Wi-38 cells, the host
cells for their herpesvirus infections, were not inhibited by phos-
phonoacetate. Huang (2) and Hirai and Watanabe (11) also reported
that the DNA polymerases a and B of Wi-38 cells were not inhibited.
In contrast, Bolden et a1. (12) showed that, although_the
B-polymerase of human HeLa cells was not inhibited by phosphono-
acetate, the a-polymerase was inhibited. Moreover, Bolden et a1.
(12) reported that the HeLa a-polymerase was as sensitive to phos-
phonoacetate as the herpesvirus-induced DNA polymerase. For duck
embryo fibroblasts, Leinbach et al. (9) demonstrated that the

a-polymerase was sensitive to phosphonoacetate, but that it was 15

l

2
to 30 times less sensitive to the inhibitor than the herpesvirus-
induced DNA polymerase.

With regard to the human cells, Overby et a1. (13) suggested
that the apparent differences in the sensitivities of the
a-polymerases from Wi-38 and HeLa cells might be explained by the
fact that the two cell types arose from different sources. Wi-38
cells originated from normal embryonic lung tissue (14) and HeLa
cells arose from cervical cancer tissue (15). They argued that
normal and transformed cells might have different a-polymerases.

In this report the effect of phosphonoacetate on the
a-polymerase of Wi-38 and HeLa cells was reexamined. The
a-polymerase of phytohemagglutinin-stimulated human lymphocytes was
also examined. In addition, the a-polymerases of Chinese hamster
ovary cells and calf thymus as well as the DNA polymerases from two
non-vertebrate eukaryotic cells, yeast and tobacco cells, were
looked at. Finally the effect of several phosphonate analogues on

the HeLa a-polymerase was investigated.

LITERATURE REVIEW

Vertebrate DNA Polymerases

 

Three general classes of DNA polymerases are found in cells of
all vertebrates: DNA polymerase a, DNA polymerase B, and DNA
polymerase y. The Greek letters indicate the historical order of
discovery of the DNA polymerases. These DNA polymerases can be
distinguished from one another by their chromatographic properties,
molecular weight, sensitivity to N-ethylmaleimide, salts, and heat,
and ability to copy various templates. Each of these enzymes has
been reported from numerous sources, as has been reviewed by Weissbach
(38,39) and Wintersberger (40). In this discussion the general

properties of the enzymes will be noted.

DNA Polymerase a

 

This class of DNA polymerase is exemplified by the calf thymus
polymerase which was purified and extensively studied by Bollum and
his co-workers (41,42). These studies provided a framework for
future studies from other origins. The a-polymerase is referred to
as the high molecular weight vertebrate polymerase which when dis-
aggregated exhibits a sedimentation coefficient of 6 to 88. The
purified enzyme from human KB cells shows a native molecular weight
of 175,000 daltons and contains an 87,000 dalton peptide subunit (43).
The a-polymerase of calf thymus is a single polypeptide of molecular

weight 155,000 to 170,000 and seems to contain an additional subunit

4

of 50,000 to 70,000 daltons (44,45). The anomalous behavior of the
a-polymerase on gel filtration is probably due to the molecule's
asymmetry. The a-polymerase is an acidic protein. The isoelectric
points of the polymerase from human lymphocytes, human KB cells, and
calf thymus are in the range of 4.5 to 5.5 (46,47,48). The activity
of a—polymerase in growing cells represents 80 to 90% of the total
DNA polymerase activity (38).

The a-polymerase has often been referred to as the "cytoplasmic
DNA polymerase" because it has been found predominantly in the cyto-
plasm when cells are subjected to normal extraction procedures.
Many investigators feel that this cytoplasmic localization is an
artifact. Lynch et a1. (49) reported that when rat liver is homogen-
ized in 0.3 M sucrose and 4 mM CaCl2 only 1.4% of the total
0-polymerase is found in the cytoplasmic fraction. If the solution
used for the homogenization contained Tris buffer and/or KCl the
a-polymerase leached out of the nucleus. When glycerol is used to
prepare the nuclei approximately 85% of the a-polymerase is in the
nucleus. Herrick et a1. (50) have shown that 85% of the a-polymerase
activity is associated with nuclei by subjecting mouse L cells to
cytochalasin-B—induced enucleation. Foster and Gurney (51) isolated
nuclei from mouse and human cells using a nonaqueous procedure of
cell fractionation in which lyophilized cells were homogenized and
centrifuged in 100% glycerol. They demonstrated that approximately
85% of the DNA polymerase activity of whole cells is in the nuclear
fraction. The remaining 15% of the activity had been lost during
fractionation.

The G-polymerase is particularly sensitive to high ionic strength

(44) but significant enzyme activity can be observed in the presence

S
of 25 mM NaCl. Reagents such as p-chloromercuribenzoate and
N-ethylmaleimide which block sulfhydryl groups strongly inhibit the
a-polymerase (38). The a-polymerase is inhibited by high concentra—
tions of actinomycin D and by low concentration of ethidium bromide
(52). The inhibition of the a-polymerase by arabinofuranosyl
cytosine-triphosphate may be due to the effect of this nucleotide
analogue on DNA synthesis (53). Pyrophosphate is an inhibitor of the
d-polymerase but only a minimal dNTP-pyrophosphate exchange reaction
occurs (42,43). The a-polymerase from human KB cells promotes an
exchange reaction which is about 0.8% of the polymerase activity.
The a-polymerase from calf thymus has been shown by Herrick et al.
(54) to be stimulated by two helix unwinding proteins isolated from
calf thymus.

No nuclease activity has been found associated with the most
highly purified a-polymerase preparations. With the a-polymerase
from human KB cells, the limit of detection for nuclease activity
was 0.003% of the polymerase activity based on production of acid
soluble radioactive nucleotide fragments (43). Employing a highly
sensitive endonuclease assay, which measured the conversion of super-
coiled SV40 DNA to SV40 DNA with single strand nicks, to assay the
most highly purified fraction of a-polymerase from regenerating
rat liver, no nuclease activity was detected.

The a-polymerase requires 3'OH priming groups as initiators
and utilizes gapped duplex DNA (activated DNA) at a high rate.
Spadari and Weissbach (55) reported that a-polymerase from human
HeLa cells can utilize DNA primed with natural RNA. The a-polymerase

cannot use synthetic ribohomopolymers as templates.

6

There have been several reports that a-polymerase is hetero-
geneous (37,48,56,64). In each case several peaks were obtained by
DEAR-cellulose or DEAE-Sephadex chromatography.

Spadari and Weissbach (57) used a rabbit antiserum to the HeLa
a-polymerase to show that the o-polymerase from CHO cells is sig-
nificantly inhibited by the antisera. Thus, in agreement with
Chang and Bollum (58), it appears that a-polymerases of various

species share some common peptide sequences.

DNA Polymerase B

 

The first recognition of a distinct nuclear DNA polymerase,
separable from the predominant cytoplasmic DNA polymerase and which
differed in its molecular weight and biochemical characteristics,
was reported in HeLa cells by Weissbach et a1. (22). Soon after
this several other investigators reported low molecular weight DNA
polymerases from other sources (59,60,61). Chang has purified the
B-polymerase to homogeneity from calf thymus chromatin (62). The
enzyme is homogeneous by acrylamide gel electrophoresis in the
presence of sodium dodecyl sulfate and by equilibrium sedimentation
centrifugation.

When disaggregated the B-polymerase exhibits a sedimentation
coefficient of 3 to 48. In KB cells, the enzyme, is a single poly-
peptide of molecular weight 43,000 to 45,000 daltons (32). In low
ionic strengths the B-polymerase forms aggregates comparable in size
to the a-polymerase (32). Consequently, a mistaken structural rela-
tionship was first reported for these two DNA polymerases. The
0- and B-polymerases have not been found to be antigenically related

(63).

7

The B-polymerase is a basic protein. Isoelectric points for
the enzyme from human lymphocytes, KB cells, calf thymus, and Novikoff
hepatoma cells are in the range of 8.5 to 9.5 (46,32,65,44). The
primary intracellular location of the B-polymerase is nuclear,
although small amounts have sometimes been found in the cytoplasm.
In growing cells it comprises about 5 to 15% of the total DNA polymerase
activity.

The sensitivity to high ionic strength varies with the enzyme
source. Activities in the presence of 0.2 M NaCl may be stimulated
0.5- to 4-fold above those in the absence of NaCl. The B-polymerase
is insensitive to sulfhydryl group inhibitors. This serves as a
distinguishing characteristic of the a- and B-polymerases. The
B-polymerase from a variety of mammalian cells is more rapidly
inactivated in vitro by elevated temperatures than the a-polymerase
(36). Purified B-polymerase is rather insensitive to adverse condi-
tions (62). For example, it is completely stable in 5 M urea and is
not inhibited by 20% ethanol or 25% acetone. No detectable nuclease
activity has been found to be associated with the B-polymerase and
consequently it does not excise misplaced primer termini (32). The
B-polymerase is inhibited by pyrophosphate but no detectable pyro-
phosphate exchange has been demonstrated (42,66).

Although the B-polymerase requires the four dNTPs for maximal
activity, it does show activity in the absence of one or more dNTPs.
This is probably a reflection of the fact that activated DNA may
contain at least 1013 3'0H termini per ug of DNA (38). The
B-polymerase utilizes activated DNA at a high rate and can also
utilize poly(rA)-oligo(dT)12-13 to a lesser degree (67). It cannot

use ribonucleotide primers.

8

A phylogenetic survey was conducted by Chang for B-polymerase
activity using low molecular weight and insensitivity to N-ethyl-
maleimide as primary identifying characteristics (68). This type of
polymerase has a widespread occurrence in multicellular animals
beginning with sponge, the most primitive animal surveyed. The
observation that the B-polymerase is absent from the nuclei of lower
eukaryotes was made with yeast (69). This type of polymerase also
has not been found in prokaryotes, protozoa, or plants.

The B-polymerase is immunologically distinct. This was demon-
strated using antibody prepared in rabbit against the partially
purified HeLa a-polymerase (63), reversing an earlier report by
Chang and Bollum (58). The absence of any relationship between the
a- and B-polymerases was confirmed using antiserum to a-polymerase
prepared in rats, in both direct neutralization assays and addition-
ally by immunoprecipitation of the a-polymerase antibody complex with
goat anti-rat immunoglobulin (70). In addition, it has been shown
that the a- and B-polymerases of chick embryo are immunologically

unrelated (71).

DNA Polymerase Y

 

The separation and initial differentiation of y-polymerase was
first described by Fridlender et a1. (72). Since then its presence
has been reported from a number of sources. Originally the HeLa
Y-polymerase was resolved into two forms by phosphocellulose and
hydroxyapatite chromatography (73), but it has since been found to
be a single form (29). The molecular weight for the HeLa
Y-polymerase, purified over 60,000-fold, ranges from 160,000 to

333,000 (31). Optimal activity with poly(rA)-oligo(dT)12-18 is in

9

the presence of 50 mM KPOq and 130 mM KCl. Under these conditions,
the y-polymerase copies poly(rA) twenty times more rapidly than
activated DNA. These assay conditions also permit a clear distinc-
tion between y- and B-polymerase, the latter of which is markedly
inhibited by phosphate at this concentration. The y-polymerase does
not utilize natural RNA (73). It is not antigenically related to
reverse transcriptase of the RNA tumor viruses (73,74). In addition,
the cellular y-polymerase and virus-induced reverse transcriptases
can be clearly separated and identified in Type C virus infected
cells (75,76). The y-polymerase is an acidic protein and requires
sulfhydryl groups for maximal activity (38). In growing cells it
comprises about 1 to 2% of the total DNA polymerase activity (57).
The apparent Michaelis constants for the dNTPs are lO-fold lower
than those of the a- and B-polymerase. The y-polymerase does not
catalyze any significant amount of pyrophosphate exchange (31).

Ito et al. (77) have isolated a nuclear membrane complex from
KB cells infected with adenovirus 2 that has some properties of an
adenovirus DNA replication complex. The major DNA in this complex,
which can synthesize viral DNA, is the Y-polymerase.

The original report from Fry and Weissbach (78) indicated that
HeLa cell mitochondria contained two DNA polymerases. One resembled
Y-polymerase and the other, designated mitochondrial DNA polymerase,
was a unique enzyme unrelated to the other cellular DNA polymerases.
Subsequently, tests for mycoplasma done under anaerobic conditions,
revealed that the HeLa cells used in those experiments were contaminated
with this organism (79). Mitochondria from rat liver and mycoplasma-

free HeLa cells were reexamined and found to contain a single DNA

10
polymerase activity which closely resembles the y-polymerase found

in the corresponding cytOplasm of these cells.

DNA Polymerase Activities in vivo

 

The attempts to gain some insight into the physiological func-
tions of the vertebrate DNA polymerases have involved analyses of
the levels of activity of the various enzymes in cells at various
stages of growth. The levels of DNA polymerase activity were
measured in mouse L cells and baby hamster kidney cells in log and
stationary phase (34,35). In both cell lines, there was a marked
rise in the a-polymerase in log phase with the levels of activity
being 5— to 12-fold higher than in stationary phase. The levels of
B-polymerase remained unchanged. In two reports the levels of
a-polymerase have been shown to increase in regenerating rat liver
18 to 30 hours post-hepatectomy (80,81). The levels of a-polymerase
48 hours post-hepatectomy were found to be increased 6— to 7-fold,
whereas B-polymerase levels remained constant.

Chang and Bollum (42) have shown that L cells which are shifting
from a stationary phase to a dividing state exhibit a rise in the
G-polymerase levels within 48 hours with no change in the B-polymerase
levels. The levels of DNA polymerase activities in synchronized
HeLa cells have also been studied. Spadari and Weissbach (57)
measured DNA polymerase activities in crude nuclear and cytoplasmic
extracts of synchronized HeLa cells. They observed a rise in
Y-polymerase in the early part of the S phase, a steady rise in
a-polymerase during the entire S phase, and no change in B-polymerase.
Chin and Baril (82) have extended this approach by examining the

complete cell cycle in synchronized HeLa cells. They measured

11
nuclear DNA polymerase activities which had.been partially purified
by phosphocellulose column chromatography. They observed a 7- to
lO-fold increase in the o-polymerase activity in the nucleus and
cytoplasm between the late 61 and mides phase and a subsequent
rapid decline between late S and GZ. No changes in the B-polymerase
levels were observed and no reproducible variation in the y-polymerase
levels were observed. Addition of cycloheximide to cultures just
prior to the 61 phase abolished the rise in a~polymerase levels,
indicating that protein synthesis was necessary for the increase in
enzymatic activity. The a-polymerase activity increased in the cell
cycle even if DNA synthesis was blocked by hydroxyurea. Since it
is known that hydroxyurea does not prevent synchronized 61 cells
from entering the S phase (83), the finding that the rise and fall
in a-polymerase activity is apparently independent of DNA synthesis
signifies there is no simple correlation between DNA synthesis and
DNA polymerase levels (39). Noy et a1. (37) have found that cyclo-
heximide leads to selective effects on the levels of the a-, B-, and
Y-polymerases. The level of the B-polymerase fell rapidly to 30%
of its initial value during the first seven hours of exposure to
cycloheximide. While the level of the a-polymerase remained unchanged
during the first seven hours, both the a- and y-polymerases dropped
to 50% of their original level after 24 hours.

In another study Bertazzoni et a1. (23) measured DNA polymerase
levels in phytohemagglutinin-stimulated, ultraviolet-irradiated
human lymphocytes. The a- and B-polymerase levels were measured in
crude extracts or after sucrose gradient centrifugation of crude
extracts. In the stimulated lymphocytes a wave of DNA synthesis was

observed which reached maximal levels after five days of stimulation

12
and later decreased to levels lower than one-tenth of the maximum.
Two levels of repair synthesis were observed. One reached maximal
levels approximately one day in advance of the peak in DNA replica-
tion rate and a second wave occurred three days later when the overall
DNA synthesis rate was in the decreasing phase. The levels of
o-polymerase paralleled the levels of DNA replication. A 20-fold
increase in enzyme activity was observed after five days of stimula-
tion. The B-polymerase levels increased more slowly and to a lesser
extent and seemed to parallel the second wave of repair synthesis.
After seven days of stimulation, maximal levels were about 7—fold
greater than those in the unstimulated cells. From these results
the investigators suggested a correlation between a-polymerase
activity and DNA replication, and between B-polymerase activity and
repair synthesis.

Thus, there is evidence from several laboratories that the
a-polymerase is one of the major proteins involved in DNA replica-
tion since a-polymerase levels change with the proliferative state
of the cell and it can use RNA—primed DNA as template. The physio-
logical functions of the B- and y-polymerases are less clear; the

B-polymerase may be involved in DNA repair synthesis.

DNA Polymerases During Development

 

Differentiating systems have been studied in an attempt to find
a pattern in the changes in DNA polymerase activity that occur as
cells become specialized or mature. Spermatogenesis is one of the
systems used to study changes in DNA polymerase during development.
Chevaillier and Philippe (84) and Hecht et al. (85) examined the

developing mouse testes. The nuclear B-polymerase activity increased

13
during meiotic prophase (84) and the orpolymerase activity diminished
during the attainment of sexual maturity (85). Sherman and Kang
(86) investigated a- and B—polymerase levels of midgestation mouse
embryo, trophoblast, and decidua. The enzyme levels were highest in
the rapidly dividing embryonic cells, significantly lower in DNA
replicating but nondividing trophoblast cells, and lowest in the
nonreplicating, nondividing decidual cells. Changes in the DNA
polymerase levels have been followed during sea urchin development
(87,88) and in Xenopus laevis (89,90,91). An increase in the polymer-
ase activity was observed as the eggs matured.

In studies on the pattern of developmental changes of DNA
polymerase in rat brain, Chiu and Sang (92) found that the
a-polymerase showed higher activity than the B-pOlymerase in rat
cerebellum immediately postnatal. Furthermore, the o-polymerase
decreased rapidly with age and was much lower than that of the
B-polymerase. The cerebellum of the rat grows rapidly for sixteen
days after birth and shows rapid cell proliferation during this
period. The DNA concentration continues to increase up to two weeks.
The investigators found that in the rat cerebral cortex the
a-polymerase showed higher activity than the B-polymerase only at
the fetal stage. The a-polymerase was low after birth and essen-
tially no activity was found in the adult. In the cerebral cortex
of the rat, cell division is almost complete at birth. The DNA
concentration decreases during maturation and the activity for DNA
synthesis is very low. Therefore, the a—polymerase showed high
activity in a proliferating stage and low activity when mitosis

ceased.

l4

Barton and Yang (93) examined the activities of o- and B-
polymerase in spleens of Balb/c mice during aging. No change in
the a-polymerase activity was noted, whereas the B-polymerase
activity was decreased in the spleens of senescent animals compared
with that of younger animals. DNA polymerase activity in normal
and malnourished rat placentas has been measured and found to correlate
with DNA synthesis. Thus, there is evidence from several laboratories
that as cells progress toward maturity the level of DNA polymerase

declines in parallel with the level of DNA synthesis.
Phosphonoacetate and Herpesvirus Infection

Initial Studies with Phosphonoacetate

Using a random testing of compounds with a tissue culture screen,
workers at Abbott Laboratories discovered that phosphonoacetate was
an effective inhibitor of the replication of herpes simplex virus
types 1 and 2 (1). These initial studies using phosphonoacetate as
an antiherpesvirus agent involved herpes simplex virus infection of
mice and rabbits. Mice were infected with RSV-2 by tOpical applica-
tion to denuded skin. The resulting herpes dermatitis caused death
in untreated mice. However, if phosphonoacetate was administered
orally or topically to infected mice two hours after infection,
mortality was significantly reduced. Rabbits were infected with
HSV-l by topical application to the eyes. The resulting herpes
keratitis caused corneal lesions in untreated rabbits. However, if
an ointment of phosphonoacetate was applied to the eyes of infected
rabbits two hours after infection, a reduction in the lesions was

observed.

15

The inhibitory effect of phosphonoacetate on herpesvirus
infections in tissue culture was first documented by investigators
at Abbott Laboratories (5). The infection of Wi-38 cells with
HSV-l resulted in rounded, clumped, and multinucleated cells. How—
ever, when HSV-l-infected Wi-38 cells were cultured in the presence
of 500 uM phosphonoacetate, cytopathic effects were not observed.
The infected cells could not be distinguished from the uninfected
cells. This suggested that phosphonoacetate prevented the‘replica-
tion of HSV-1.

Studies were also performed to determine at what stage of
infection phosphonoacetate inhibited (5). Phosphonoacetate appeared
to have no effect on absorption, penetration, or release of the
virus. When phosphonoacetate was removed from the culture medium
of HSV-1 infected Wi-38 cells, cytOpathic effects reappeared. Thus,
the viral genomes were still present and functional in the infected
cells. No difference in RNA and protein synthesis was observed
between phosphonoacetate-treated and untreated infected cells, but ‘
a reduction in DNA synthesis was noted in the phosphonoacetate-
treated infected cultures. The inhibition of DNA synthesis was
specific for viral DNA synthesis as determined by isolation and
bouyant density centrifugation of DNA from infected cultures grown
in the presence and absence of phosphonoacetate. Twelve hours post-
infection two peaks of [1“C1thymidine, corresponding in density to
HSV-l DNA and cellular DNA were found in CsCl gradients for untreated
cells. Only one peak of [;”C]thymidine, corresponding in density to
cellular DNA, was found in CsCl gradients for phosphonoacetate-treated

cells.

l6

Overby et al. (5)7were also interested in the effect of phos-
phonoacetate on uninfected cells. Phosphonoacetate was not toxic
to contact inhibited Wi-38 cells. No morphological changes were
observed when confluent Wi-38 cells were incubated in the presence
of 1 mM phosphonoacetate for two days. No effect was noted on DNA,
RNA, or protein synthesis. However, phosphonoacetate was toxic to
growing cells. When 500 uM was present in the culture medium a 40%
decrease in the number of cells in culture was observed, whereas
no toxic effects were noted when 50 uM phosphonoacetate was present
in the culture medium.

Mao et a1. (6) first demonstrated that the inhibition of herpes-
virus replication by phosphonoacetate is through an effect on the
herpesvirus-induced DNA polymerases. When HSV-l-induced DNA polymerase
was assayed in the presence of 1 uM phosphonoacetate, enzyme activity
was inhibited by 50%.

Phosphonoacetate Inhibition of Herpes-
viruses and Other Viruses

 

Since the initial studies of Shipkowitz et a1. (1) and Overby
et al. (5) which demonstrated the effectiveness of phosphonoacetate
in inhibiting the replication in cell culture of herpes simplex
viruses, other herpesviruses have been found to be sensitive to
phosphonoacetate. At a concentration of 500 uM, phosphonoacetate
inhibited the replication of the avian herpesviruses, Marek's disease
herpesvirus, herpesvirus of turkeys, and owl herpesvirus (3). Viral
DNA synthesis as determined by CsCl equilibrium density gradient
centrifugation or by DNA-cRNA hybridization was significantly
reduced in viral-infected cells treated with phosphonoacetate. Inhi-

bition of viral replication and DNA synthesis was also Observed with

17
three types of cytomegalovirus, human, simian, and murine (2,95).
Viral DNA synthesis resumed when phosphonoacetate was removed, indi-
cating that the inhibition was reversible. Similar results were
noted with herpesvirus saimiri in owl monkey kidney cells. Phosphono-
acetate inhibited herpesvirus saimiri and the synthesis of virus-
induced intracellular late antigens and membrane antigens, but not
the synthesis of early antigens (96). The continued presence of
phosphonoacetate was required to suppress replication (97). For
example, maintenance of the infected cells on phosphonoacetate for
63 days had no effect on completely suppressing replication after
removal. Phosphonoacetate also inhibited the replication of equine
abortion herpesvirus (13,98), Epstein-Barr virus (4,10,99), and
varicella zoster virus (13) in culture by inhibiting viral DNA
synthesis. Although phosphonoacetate appears to be a universal
inhibitor of herpesvirus DNA synthesis, inhibition has also been
demonstrated for vaccinia virus to a lesser extent (13). The three
RNA viruses, poliovirus, rhinovirus, and measles virus, are not sig-
nificantly sensitive (13).

Phosphonoacetate blocked herpes simplex virus DNA synthesis in
nuclei prepared from infected cells (12,100). In addition, an
inhibition by phosphonoacetate of in vitro DNA synthesis by nuclei
from Raji cells superinfected with EBV was noted (101). Both cellular
and viral DNA synthesis were inhibited, although synthesis of cellular
DNA may have been inhibited to a somewhat lesser degree than synthesis

of EBV DNA.

18

Phosphonoacetate Inhibition of the
Herpesvirus-Induced DNA Polymerase

 

 

Phosphonoacetate was a specific inhibitor of herpes simplex
virus-induced DNA polymerase (6). The sensitivity of the herpesvirus
type 1 and 2 DNA polymerases was similar. The Ki values for the
HSV—2-induced DNA polymerase were 0.45 to 0.47 uM (7). Phosphono-
acetate did not inhibit the herpesvirus-induced DNA polymerases
because of the formation of a phosphonoacetate-DNA complex (2,6).

The specificity of phosphonoacetate for the polymerase precludes

this possibility. Moreover, phosphonoacetate inhibition was not
decreased when DNA concentrations were increased in the DNA polymeri-
zation reaction mixtures. Finally, the presence of phosphonoacetate
in HSV-l-induced polymerization reaction mixtures did not prevent
formation of an enzyme-DNA complex as determined by glycerol density
gradient sedimentation velocity centrifugation of these reaction
mixtures (6). Phosphonoacetate was an effective inhibitor of both
the Marek's disease herpesvirus and the herpesvirus of turkeys-induced
DNA polymerase (9). The phosphonoacetate inhibition patterns and
apparent inhibition constants for the DNA polymerization reaction
catalyzed by the herpesvirus of turkeys-induced DNA polymerase were
determined. Phosphonoacetate gave linear noncompetitive inhibition
with the four dNTPs as the variable substrate and activated DNA at

a saturating concentration of 200 ug/ml. With activated DNA as the
variable substrate, and the four dNTPs at their apparent Michaelis
constant concentrations of 2.5 uM each, phosphonoacetate gave linear
noncompetitive inhibition with activated DNA as the variable sub-
strate and the four dNTPs at 100 uM each. The apparent inhibition

constants were 1 to 2 uM. Phosphonoacetate was then shown to be a

l9
competitive inhibitor of pyrophosphate in the dNTP-pyrophosphate
exchange reaction. Multiple inhibition analysis showed that phosphono-
acetate and pyrophosphate were mutually exclusive inhibitors. When
taken together, the above results showed that phosphonoacetate
inhibited the polymerase by interacting with it at the pyrophosphate
binding site and functioned either as a dead-end inhibitor or as an
alternate product (9, J. Boezi, personal communication).

Other herpesvirus-induced DNA polymerases have been shown to be
inhibited by phosphonoacetate. The human cytomegalovirus-induced
DNA polymerase (2,11), equine herpesvirus-induced DNA polymerase
(98), and EBVeinduced DNA polymerase (102) have all been shown to
be sensitive.

Studies of phosphonoacetate-resistant mutants of herpesvirus
support the hypothesis that phosphonoacetate inhibits herpesvirus
replication through a specific inhibition of the herpesvirus-induced
DNA polymerase. Hay and Subak-Sharpe (103) isolated mutants of HSV
which formed plaques in the presence of 100 ug/ml phosphonoacetate.
Three of the mutants induced viral DNA synthesis and viral DNA
polymerase activity which was much less sensitive to phosphonoacetate
than the wild type virus. Similar results were observed by Becker
et al. (100) with HSV mutants isolated from S-bromodeoxyuridine-
mutagenized virus that were resistant to phosphonoacetate at high
concentrations. Honess and Watson (104) passed the virus in the
presence of phosphonoacetate to select resistant mutants. Stable
clones of mutant viruses with a range of drug sensitivities were
isolated. A higher level of drug resistance could be correlated

with increased resistance of virus DNA synthesis, y-protein synthesis

20
(late virus proteins), and resistance of the virus-induced DNA
polymerase to the inhibitory effects of phosphonoacetate.

Studies of the analogues of phosphonoacetate indicate the
structural requirements for antiherpesvirus action are rather
narrowly defined (1,3,9,105, Reno et al., submitted for publication).
Leinbach et al. (9) observed that 2-phosphonopropionate was an
inhibitor but that the apparent inhibition constant for it was about
50 times greater than the corresponding apparent inhibition constant
for phosphonoacetate. Herrin et al. (105) noted that propyl phos-
phonoacetate was as effective as phosphonoacetate in inhibiting the
HSV-induced DNA polymerase and in animals with herpes dermatitis.
The inhibitory effect of phosphonoformate was recently discovered
by Reno et al. (submitted for publication). This compound was as
effective as phosphonoacetate in inhibiting the herpesvirus of
turkey-induced DNA polymerase. The inhibition constants and inhibi-
tion patterns were identical to those reported for phosphonoacetate.
Phosphonoformate also inhibited the replication of Marek's disease
herpesvirus, the herpesvirus of turkeys, and herpes simplex virus.

Effect of Phosphonoacetate on
Other DNA Polymerases

 

 

The sensitivity of other DNA polymerases to phosphonoacetate
has been examined. The o-polymerase from Wi-38 cells has been
reported to be relatively resistant to phosphonoacetate inhibition
(2,6,7,11). ,When the Wi-38 a-polymerase, partially purified by
phosphocellulose chromatography, was assayed in the presence of 300
uM phosphonoacetate, only 10 to 15% inhibition of enzyme activity
resulted (6). In contrast, Bolden et al. (12) found that the

a-polymerase from.HeLa cells was inhibited by 50% when assayed in

21
the presence of 30 pH phosphonoacetate. Phosphonoacetate was also
an inhibitor of the a-polymerase from HEL (human embryonic lung)
cells (33) and duck embryo fibroblasts (9) at concentrations which
are 15 to 30 times higher than those which inhibit the herpesvirus-
induced DNA polymerases. The B-polymerase from a number of cells has
been found to be relatively insensitive to phosphonoacetate (2,6,7,
9,11,12,33), as has the y-polymerase from HeLa cells (29).

The vaccinia virus-induced DNA polymerase was inhibited by 50%
when assayed in the presence of 30 uM phosphonoacetate (12). Other
viral DNA polymerases such as the reverse transcriptase from avian
myeloblastosis virus (9) and Rous sarcoma virus (7), and hepatitis B
virus DNA polymerase (7) were insensitive to phosphonoacetate. The pro-
karyotic DNA polymerases, Escherichia coli DNA polymerase I (9), Micro-
coccus luteus DNA polymerase (7), and the 68 mycoplasma DNA polymerase (33,
J. Boezi, personal conmunication) are. not inhibited by phosphonoacetate.

At concentrations of 100 ug/ml or less phosphonoacetate has been
shown to have no obvious effect on cell growth and cellular DNA
synthesis in Wi-38 cells (2,5), lymphocytes carrying the EBV genome
(4,10), or duck embryo fibroblasts (3). Phosphonoacetate at concen-
trations of 500 to 1000 ug/ml causes inhibition of growth and cellular
DNA synthesis, an increase in the diameter of the cell, and probably
causes an arrest at interphase.

Effect of Phosphonoacetate on a Cell Line
Transformed by Epstein-Barr Virus

 

 

Epstein-Barr virus is a herpesvirus for which no completely per-
missive culture system exists, i.e., no cell type supports the
efficient replication of EBV. There are, however, several well

characterized human lymphoblastoid cell lines transformed by EBV.

22
These cell lines have been cultured in the presence of phosphono-
acetate in an attempt to determine the mechanism of EBV DNA
replication (4,99).

The Raji cell line is a transformed lymphoblastoid cell line
derived from a tumor biopsy from a patient with Burkitt's lymphoma
(106). It is a nonproducer cell line. Raji cells contain approxi-
mately 50 EBV genomes per cell but express only a single viral
antigen, the EBV-associated nuclear antigen, and produce no viral
particles. Replication of resident EBV genomes is under cellular
control mechanisms and occurs only during early S phase (107). If
Raji cells are superinfected with EBV, host cell DNA synthesis is
completely inhibited and productive replication of EBV occurs (99).

The effect of phosphonoacetate on the EBV content in Raji cells
before and after superinfection has been determined. When Raji cells
were grown in the presence of 1 mM phosphonoacetate for three days
or in the presence of 0.5 mM phosphonoacetate for three weeks,
there was no change in the number of resident EBV genomes as determined
by hybridization techniques (4,99). Therefore, the EBV DNA present
in these transformed cells replicates by a mechanism which is insen-
sitive to phosphonoacetate as might be expected since resident EBV
DNA replication is under cellular control.

When Raji cells were superinfected, the amount of EBV DNA present
in these cells increased to such levels that it could be detected in
CsCl gradients if the superinfected cells were labeled with [3H1thymi-
dine (99). When Raji cells were superinfected and then cultured in
the presence of 0.5 liphosphonoacetate, EBV DNA could not be
detected in CsCl gradients. Therefore, productive EBV DNA replica-

tion in the absence of cellular controls is sensitive to

23
phosphonoacetate. This was further demonstrated in D98/HR-l cells
(102). Under normal growth conditions the only EBV-specific antigen
detectable in D98/HR-l cells by the immunofluorescence assay is the
EBV-associated nuclear antigen as in Raji cells. S-Iodo-2'-deoxy-
uridine induces EBV-specific early antigen, viral capsid antigen,
and membrane antigen, and DNA synthesis is initiated) the cells
therefore resemble producer cells. The effect of phosphonoacetate
on the induction of EBV antigens was determined by adding phosphono-
acetate at the time of removal of IUdR. Phosphonoacetate (100 ug/ml)
had little effect on the expression of EBV-specific early antigen,
but markedly reduced the expression of the viral capsid antigen.
This result suggests that an EBV-specific DNA polymerase might be
induced by IUdR since the viral capsid antigen is dependent specifi-
cally upon viral DNA synthesis (108). Miller et al. (102) identi-
fied a salt-stimulated DNA polymerase in D98/Her cells which
resembled other herpesvirus-induced DNA polymerases.

The 895-8 cell line is a marmoset cell line derived by treatment
with EBV from a human cell line established from a patient with acute
infectious mononucleosis believed to be induced after blood trans-
fusion (109). B95-8 cells release infectious virus particles and
contain about 150 EBV genome equivalents per cell. The three virus
specific antigens, EBV-associated nuclear antigen, early antigen, and
viral capsid antigen, are expressed. The synthesis of EBV in B95-8
cells is completely inhibited by phosphonoacetate, as shown by the
total inhibition of viral capsid antigen synthesis. This effect of
phosphonoacetate on 895-8 cells and P3Her cells, a human lympho-
blastoid cell line, was also studied by Nyormoi et al. (10).

Thorley-Lawson and Strominger (110) infected normal human peripheral

24
B lymphocytes with the transforming strain of EBV from the super-
natant of a 895-8 marmoset lymphocyte culture. The B lymphocytes
became a transformed cell line. Phosphonoacetate at a concentration
of 100 to 200 ug/ml inhibited the transformation and the outgrowth
of the EBVbinfected cultures. Taken together, the above results are
consistent with the hypothesis that EBV DNA is replicated by two
mechanisms, one in the noninduced cell which is insensitive to
phosphonoacetate and a different mechanism in the producer cell
which is sensitive to phosphonoacetate. This second mechanism has
been shown to involve an EBV-induced DNA polymerase which resembles

the herpesvirus-induced DNA polymerases (102).

Animal Studies with Phosphonoacetate

 

The first studies using phosphonoacetate as an antiherpesvirus
agent in animals, as reported by Shipkowitz et al. (1), involving
herpes simplex virus infections of mice and rabbits, were previously
discussed. Further studies in animal model systems demonstrate the
efficacy of phosphonoacetate as an antiherpesvirus drug. The effec-
tiveness of phosphonoacetate in the treatment of herpes dermatitis
in mice was confirmed (lll). Hairless mice infected percutaneously
with inhibitor-resistant or with parental inhibitor-susceptible HSV
strain were treated intraperitoneally with phosphonoacetate. Only
the infection induced by the parental phosphonoacetate-sueceptible
virus was suppressed by phosphonoacetate. Both the phosphonoacetate-
resistant and the phosphonoacetate-susceptible HSV-induced infections
were suppressed by 9-B-D—arabinofurabosyl-adenine.

While the experimental model used by Abbott Laboratories in

treating herpes keratitis was useful for screening purposes, it bore

25

little relationship to the problems faced in treating established
herpes keratitis (112). To test the effect of phosphonoacetate and
S-iodo-2'-deoxyuridine (the only drug comercially available to
treat herpetic keratitis) on established HSV corneal infections in
rabbits, treatment was started three days postviral inoculation and
was continued for five days. The effect of topically applied anti-
viral agent in one eye was compared to no treatment or placebo in
the other eye of each animal. The effect of a 5% phosphonoacetate
ointment was equivalent to that of a 0.5% S-iodo-Z'deoxyuridine
ointment in the treatment of established herpetic eye infection.
Similar studies were done to test the effect of phosphonoacetate
against deep ocular herpetic infections (113). Treatment started
three days after infection and continued for five days. Phosphono-
acetate had a significant antiviral effect when applied tapically
in both liquid and ointment preparations on superficial herpes
keratitis. Phosphonoacetate was not effective in the treatment of
experimental herpes iritis when applied topically but was signifi-
cantly effective when administered intravenously and subconjunctivally.

Genital infection of mice with herpesvirus hominis type 2 pro-
vides an experimental model for the testing of the antiviral potential
of phosphonoacetate (114). Intravaginal treatment with phosphono-
acetate (500 mg/kg in saline or as a 5% cream) initiated three hours
after inoculation with herpesvirus hominis type 2 completely inhibited
viral replication in the genital tract and prevented subsequent
mortality. Therapy initiated 24 to 72 hours after infection signifi-
cantly reduced titers of virus in vaginal secretions, but most mice
eventually died of encephalitis. Treatment intraperitoneally had no

effect on local viral replication or final mortality.

26

The antiviral activity of phosphonoacetate on mice with herpes
encephalitis caused by HSV type 1 was investigated (115). The
criteria used for judging were the rate of survival and the concen-
tration of virus in the brain. Phosphonoacetate administered subcue
taneously resulted in the long-term survival rate of approximately
15% of the infected, treated animals with four days of treatment.

When treatment was extended to seven days, the overall survival rate
was increased to 35%. There was also a reduction in the titer of
virus in the brain with treatment. The ability of a topical applica-
tion of phosphonoacetate after inoculation with HSV to prevent
infection of dorsal root ganglia in mice, and whether such applica-
tion would have an effect on established latent ganglionic infection,
were also investigated (116). When administered 24 hours after HSV
inoculation, phosphonoacetate markedly reduced skin lesions, mortality,
and the development of a latent ganglionic infection. When given
after 24 hours, phosphonoacetate reduced skin lesions and mortality
but had little effect in preventing infection.

Phosphonoacetate was effective in the treatment of mice with
lethal cytomegalovirus infections (117). Treatment was with 250 mg/kg
of phosphonoacetate injected intraperitoneally twice daily for seven
days. A marked reduction in viral titers in the brains and lungs of
infected mice was observed. The major reason for efficacy appeared
to be a complete inhibition of viral replication in the liver. Treat-
ment of nonlethal cytomegalovirus infection with phosphonoacetate
beginning 48 hours after viral challenge resulted in elimination of
clinical signs of illness and reduction in viral titers in tissue.

Chicks were infected with Marek's disease herpesvirus after

seven days of treatment with phosphonoacetate at a dosage level of

27
500 mg/kg/day (118). The birds were treated for an additional
fourteen days and observed daily for paralysis and death. The
incidence of paralysis in non-treated chicks was found to be four
times greater than that in treated birds, and the first signs of
paralysis occurred five days earlier in the non-treated chicks than
in the treated. However, phosphonoacetate was not effective in
inhibiting the replication of Marek's disease herpesvirus (3).

The efficacy of phosphonoacetate in the treatment of vaccinia
virus has been investigated (119). The effectiveness was evaluated
by the ability of phosphonoacetate to suppress the formation of tail
lesions in mice. Other investigators studied the antiviral efficacy
of phosphonoacetate in localized skin lesions of rabbits produced by
the intradermal inoculation of vaccinia virus and of Shope fibroma
virus (120). Complete suppression of the appearance of vaccinia
virus-induced pustular lesions was achieved by a 2% phosphonoacetate
ointment applied twice daily for four days. Phosphonoacetate was
significantly effective against Shope fibroma virus-induced tumors
when applied as an ointment beginning either 24 or 72 hours after
virus inoculation.

The studies on toxicity and drug metabolism for phosphonoacetate
have been carried out by Abbott Laboratories although little has
been published. Phosphonoacetate was not mutagenic in the Salmonella
typhimurium test (121). When administered orally, phosphonoacetate
was poorly absorbed in rat, rabbit, and monkey as determined by
urinary excretion (122). Absorption was somewhat higher in dogs but
varied. When administered intravenously, a substantial amount of

phosphonoacetate remained in the body. Whole body autoradiography

28
studies in rats demonstrated that it accumulated in bone and that
the retention was long term.

Abbott Laboratories has recently applied for permission to have
phosphonoacetate classified as an investigative drug for purposes of
initiating clinical trials. The application was made to the Food
and Drug Administration. Based on the toxicity of phosphonoacetate,
the FDA did not sanction usage and requested Abbott Laboratories to
undertake an additional series of toxicity tests. The results of
these tests have not been reported. Therefore, the prospective

clinical use of phosphonoacetate remains uncertain.

EXPERIMENTAL PROCEDURES

Reagents

Phosphonoacetic acid was from Richmond Organics. Trisodium
phosphonoformate and triethyl phosphonoformate were prepared follow-
ing the procedure of Nylen (16). Deoxythymidine 5' phosphophosphono-
acetate was a gift from Dr. Susan S. Leinbach, formerly of this
laboratory. Phosphonoacetamide and N-methylphosphonoacetamide were
gifts from Dr. George Stark, Stanford University. Acetonyl phosphonate
was a gift from Dr. Ronald Kluger, University of Toronto. The
o-polymerase from calf thymus and the y-polymerase from fetal calf
liver were obtained from Worthington. Other reagents were from

sources previously described (9) or from the usual commercial sources.

Cell Cultures

 

HeLa (CCL 2) and Wi—38 (CCL 75) cells were purchased from the
American Type Culture Collection. Chinese hamster ovary cells (CHO),
proline requiring, were obtained from Dr. Louis Siminovitch, University
of Toronto. HeLa, Wi-38, and CHO cell cultures were tested at 37°C
aerobically and anaerobically for myc0plasma contamination as
described by Hayflick (l7) and were not found contaminated. HeLa
and CHO cells were grown in suspension culture in F-l4 and F-20
medium (Gibco), respectively, supplemented with 10% fetal calf serum.
Wi-38 cells (passages 16-29) were grown in monolayer culture in G-13

medium (Gibco) supplemented with 10% fetal calf serum. Saccharomyces

29

30
cerevisiae, x2180 diploid strain, was obtained from Dr. William L.
Smith of this department and was grown in glucose-yeast extract-
casamino acid medium. Tobacco XD cells (Nicotiana tabacum L. var.
Xanthi) were a gift from Dr. Philip Filner of this department and
were grown according to the procedure of Filner (18). All cells were
harvested in mid log phase and frozen at -70°C until use. Lympho-
cytes were purified from human blood by centrifuging over Ficoll-
Paque (Pharmacia) (19). The lymphocytes were diluted in G-l3 medium
containing 10% fetal calf serum and 0.8% (v/v) phytohemagglutinin-P

(Difco) and cultured for 4.5 days.

Assay of the DNA Polymerization Reaction

The standard reaction mixture for the a-polymerase contained
in 200 pl: 50 mM Tris-hydrochloride (pH 8.0), 1 mM dithiothreitol,
500 ug/ml bovine serum albumin, 10 mM MgC12, 20 mM KCl, 200 ug/ml
of activated calf thymus DNA (DNase I treated), 20 uM 3H-labeled
deoxyribonucleoside triphosphate, 100 uM each of the other three
deoxyribonucleoside triphosphates, and o-polymerase. For the
B-polymerase and the yeast DNA polymerases, KCl was omitted from
the standard reaction mixture. For the tobacco cell DNA polymerase,
KCl was omitted, dithiothreitol was 20 mM, and MgClg was 6 mM. The
standard reaction mixture for the fetal calf liver y-polymerase
contained in 200 ul: 50 mM Tris-hydrochloride (pH 8.0), 2.5 mM
dithiothreitol, 500 ug/ml bovine serum albumin, 4 mM MgC12, 100 mM
KCl, 50 ug/ml poly(rA)-oligo(dT)12-13, 100 uM 3H—labeled dTTP, and
y-polymerase. Incubation was at 37°C for 30 min. Assay of the
conversion of 3H-labeled deoxyribonucleoside triphosphate into a

trichloroacetic acid insoluble form was as previously described (20).

31
Assay conditions were used so that the rate of DNA polymerization
was linear with time and with the amount of DNA polymerase. For the
kinetic studies, changes in the concentrations of assay components

are as noted in the legends to the figures.

Purification of the DNA Polymerases

HeLa and CHO cells were fractionated into a nuclear and cyto-
plasmic fraction according to the procedure of Chang et a1. (21).
The purification of the o-polymerase from the cytoplasmic fraction
and the B-polymerase from the nuclear fraction was as described in
the procedure of Weissbach et al. (22). This procedure involved
purification by DEAE cellulose and phosphocellulose column chroma-
tography. The peak fractions of DNA polymerase activity from the
phosphocellulose column were pooled, made 50% (v/v) in glycerol,
and stored at -20°C. The specific enzymatic activity (nmol of
deoxyribonucleoside triphosphate incorporated into an acid insoluble
form in 30 min at 37°C per milligram protein) was 2400 for the HeLa
a-polymerase and 1300 for the CHO a-polymerase. The purified
o-polymerases showed a single peak of enzymatic activity at about
7.58 in a linear 20 to 40% glycerol gradient containing 0.45 M KCl.
The Wi-38 o-polymerase was purified as above except that the phospho-
cellulose chromatography step was omitted. Glycerol gradient cen—
trifugation was used to purify the a-polymerase from phytohemagglutinin-
stimulated lymphocytes as described by Bertazzoni et al. (23). DNA
polymerases A and B from.S. cerevisiae were isolated through DEAE
cellulose chromatography according to Wintersberger and Wintersberger

(24). The DNA polymerase from tobacco cells was prepared as described

32
by Srivastava (25) and was further purified by DEAE cellulose

chromatography.

Inhibition Patterns
Inhibition patterns and kinetic constants were defined according
to the nomenclature of Cleland (26,27). Analysis of each reaction
mixture was done in duplicate. The data for the double reciprocal
plots were evaluated using a computer program.based on the method of
Wilkinson (28). For evaluation of the apparent inhibition constants,
replots of the intercepts and slopes of the double reciprocal plots

were analyzed using a computer program for least-squares analysis.

RESULTS

Phosphonoacetate Inhibition of the DNA Polymerization
Reaction Catalyzed bkaeLa DNA Polymerase a

 

 

Phosphonoacetate was an inhibitor of the DNA polymerization
reaction catalyzed by the HeLa o-polymerase. The addition of 25 to
30 uM phosphonoacetate to the standard reaction mixture resulted in
a decrease in the rate of the DNA polymerization reaction by about
50%. Either in the presence or absence of phosphonoacetate, the
rate of the reaction was linear for at least one hour. Phosphono-
acetate gave linear noncompetitive inhibition with the four dNTPs
as the variable substrate and activated DNA at a saturating concen-
tration of 200 ug/ml (Figure l). The apparent inhibition constant
(Kii) determined from the replot of the vertical intercepts against
the phosphonoacetate concentration was 29 uM. The apparent inhibi-
tion constant (Kis) determined from the replot of the slopes against
phosphonoacetate concentration was 53 mM. With activated DNA as the
variable substrate and the 4 dNTPs at their apparent Michaelis
constant concentrations of 7 uM each, phosphonoacetate gave linear
noncompetitive inhibition (data not shown). A replot of the vertical
intercepts yielded a Kii of 34 pH and a replot of the slopes yielded
a Kis of 94 uM. Phosphonoacetate also gave linear noncompetitive
inhibition with activated DNA as the variable substrate, the three

dNTPs at 100 uM each, and the 3H-labeled dTTP at 20 pH (Figure 2).

33

34

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35

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37

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38
The Kii for phosphonoacetate was determined to be 29 uM and the Kis
200 mM.

The above results therefore demonstrate that the HeLa a-polymerase
is indeed sensitive to phosphonoacetate. It is, however, less sensi-
tive than the herpesvirus-induced DNA polymerase. The apparent
inhibition constants for the herpesvirus-induced DNA polymerase were
in the l to 2 pH range (7,9).

Phosphonoacetate Inhibition of the DNA Polymerization
Reaction Catalyzed by Wi-38 DNA Polymerase a

 

 

Contrary to the results published by Mao et al. (6), Mac and
Robishaw (7), Huang (2), and Hirai and Watanabe (ll), phosphono-
acetate was an inhibitor of the Wi-38 o-polymerase. The inhibition
constants and the inhibition patterns were similar to those of the
HeLa a-polymerase. Phosphonoacetate gave linear noncompetitive
inhibition with the four dNTPs as the variable substrate and activated
DNA at a saturating concentration of 200 ug/ml. The Kii was 15 uM
and the Kis was 25 mM. With activated DNA as the variable substrate,
the three dNTPs at 100 uM each, and the 3H-labeled dTTP at 20 pH,
phosphonoacetate gave linear noncompetitive inhibition (Figure 3).
The Kii was 33 HM and the Kis was 150 uM.

The Effect of Phosphonoacetate on the DNA Polymerization
Reaction Catalyzed by Other a-Polymerases

 

The o—polymerase from a third human cell source was examined
for its sensitivity to phosphonoacetate. The a—polymerase from
phytohemagglutinin-stimulated lymphocytes was as sensitive as the
o-polymerase from HeLa and Wi-38 cells. Indeed, the inhibition by
phosphonoacetate seems to be a general characteristic of o-polymerases.

For example, the CHO a-polymerase and the one from calf thymus were

39

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40

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41
inhibited. For the CHO o-polymerase the inhibition patterns and
apparent inhibition constants were examined and found to be the
same as those reported above for the HeLa o-polymerase.
The Effect of Phosphonoacetate on the DNA Polymerization
Reaction Catalyzed by Other DNA Polymerases

Initial results by Mao et al. (6), Bolden et al. (12), and
Leinbach et al. (9) showed that the B-polymerase of eukaryotic cells
was not significantly inhibited by phosphonoacetate. The addition
of 200 uM phosphonoacetate to the standard reaction mixture produced
no significant inhibition in the rate of the DNA polymerization
reaction catalyzed by the HeLa or CHO B-polymerase. The B-polymerases
of Wi-38 cells and human lymphocytes were also insensitive to phos-
phonoacetate. Likewise, the Y-polymerase of fetal calf liver was
not significantly inhibited. The HeLa Y-polymerase had previously
been shown by Knopf et al. (29) not to be significantly sensitive to
phosphonoacetate.

The DNA polymerases from two non-vertebrate eukaryotic cells
were examined for their susceptibility to phosphonoacetate. The DNA
polymerases A and B from S. cerevisiae were inhibited. The rate of
DNA synthesis for either DNA polymerase A or B was decreased 50% by
the addition of 15-20 HM phosphonoacetate to the standard reaction
mixture. Similarly, the DNA polymerase from tobacco cells was sensi-
tive to phosphonoacetate at about the same concentration.

The Effect of Other Phosphonate Compounds On the DNA

Polymerization Reaction Catalyzed by
HeLa DNA Polymerase a

 

Recently it was discovered by Reno et al. (submitted for publi-

cation) that phosphonoformate is an effective inhibitor of the

42
herpesvirus-induced DNA polymerase. Phosphonoformate is also an
inhibitor of the HeLa o-polymerase. With the four dNTPs as the
variable substrate and activated DNA at a saturating concentration
of 200 ug/ml, phosphonoformate, like phosphonoacetate, gave linear
noncompetitive inhibition (Figure 4). A replot of the vertical
intercepts yielded a Kii of 24 uM and a replot of the slopes a Kis
of 59 uM. Phosphonoformate gave linear noncompetitive inhibition
with activated DNA as the variable substrate and the four dNTPs at
their apparent Michaelis constant concentrations of 7 uM each. A
replot of the vertical intercepts gave a Kii of 32 HM and a replot
of the slopes a Kis of 176 UM (data not shown). Phosphonoformate,
therefore, appears to be equally as effective as phosphonoacetate
in its inhibition of the HeLa a-polymerase.

Reno et al. (submitted for publication) have shown that phos-
phonoformate inhibits the herpesvirus-induced DNA polymerase in a
manner analogous to that of phosphonoacetate. This also seems to be
the case for the HeLa a-polymerase. A multiple inhibition analysis
(30,31) of the DNA polymerization reaction indicated that phosphono-
formate and phosphonoacetate are mutually exclusive inhibitors and,
therefore, bind at the same site on the o-polymerase. For the
multiple inhibition analysis the concentration of phosphonoformate
was varied in the presence of fixed concentrations of phosphonoacetate.
As shown in Figure 5, a plot of l/v against phosphonoformate concen-
tration resulted in a series of parallel lines, indicating the two
phosphonate compounds are mutually exclusive inhibitors.

Other phosphonate compounds produced no significant inhibition
of the rate of the polymerization reaction catalyzed by the HeLa

G-polymerase when tested to a concentration of 200 uM. The compounds

 

 

43

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on» me wnBzo “now on» nuw3 muon Heooumaoon mannoa .v ounmam

 

44

7:2 1 FT? a;

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45

Figure 5. Multiple inhibition of the HeLa a-polymerase by
phosphonoacetate and phosphonoformate. Activated DNA was at
200 ug/ml and the four dNTPs were at 7 uM each. Phosphonoacetate
concentrations were 0 (0), 10 uM (O), 20 pm (0), and 30 uM (A).

 

46

 

 

0.005

 

0.0| '- "‘

 

 

IO 20
[Phosphonoformate],( uM)

Figure 5

30

47
which were tested were imidodiphosphate, methylene diphosphate,
sulfoacetate, 2-phosphonopr0pionate, 3-phosphonopropionate, 2-
phenylphosphonoacetate, deoxythymidine 5'phosphophosphonoacetate,
trimethyl phosphonoacetate, triethyl phosphonoacetate, triethyl

phosphonoformate, phosphonoacetamide, N-methyl phosphonoacetamide,

and acetonyl phosphonate.

DISCUSS ION

This report demonstrates that the a-polymerases from three
human cells, HeLa, Wi-38, and phytohemagglutinin-stimulated lymphoé
cytes, are inhibited by phosphonoacetate. The apparent inhibition
constants (Kii) are about 30 uM. The HeLa a-polymerase has previously
been shown by Bolden et al. (12) to be inhibited by phosphonoacetate.
This report confirms their result. In addition, the apparent inhi-
bition constants and inhibition patterns for the o-polymerase are
reported. In contrast to the results of Bolden et al. (12), who
claimed that the o-polymerase and the herpesvirus-induced DNA
polymerase were equally sensitive to phosphonoacetate, the results
demonstrate that the o-polymerase is, in fact, 15 to 30 times less
sensitive.

The results showing that the Wi-38 o-polymerase is sensitive to
phOSphonoacetate contradict the results reported by Mao et al. (6),
Mac and Robishaw (7), Huang (2), and Hirai and Watanabe (11). Their
reports are in error either because their putative a-polymerase
preparations were contaminated with a myc0plasma DNA polymerase or
because their putative a-polymerase preparations were, in fact, not
a—polymerase preparations but possibly B-polymerase preparations
(32). Mycoplasma, a common contaminant of eukaryotic cell cultures,
produces a 68 DNA polymerase which is not inhibited by phosphono-
acetate (33, J. Boezi, personal communication). The 68 myc0plasma
polymerase could easily be confused with the 6-88 o-polymerase.

48

49
Indeed, the biphasic curves relating polymerase activity to phos-
phonoacetate concentration reported by Huang (2) could be interpreted
to mean that his a-polymerase preparation contained two polymerase
activities, one sensitive to phosphonoacetate which could be called
the authentic Wi-38 a-polymerase and one insensitive to phosphono-
acetate which would be labeled a possible myc0plasma contaminating
activity.

The amount of a-polymerase found in a cell is highly dependent
on the growth state of the cell (34,35). Extracts prepared from
actively growing cells contain a large amount of a-polymerase
activity and, under these growth conditions, the a-polymerase is
the major polymerase activity found in the extracts. Extracts pre-
pared from non-growing cells contain little or no o-polymerase
activity and, under these growth conditions, the B-polymerase is
the predominant polymerase activity of the extracts. Hirai and
Watanabe (11) prepared their polymerase extracts from cells that
had been mantained at confluency for 24 hours. Mao and Robishaw
(7) used an unfractionated extract in their studies. Possibly, in
contrast to their assumption, the predominant polymerase activity in
their extract was B-polymerase rather than e-polymerase.

In addition to the o-polymerase from HeLa which is a continuous
cell line that originated from cervical cancer tissue (15) and the
o-polymerase from Wi-38 which is a human cell line with a limited
capability to remain in culture and which originated from normal
human embryonic lung tissue (14), the phosphonoacetate-sensitivity
of the a-polymerase of human lymphocytes was.checked. As was the
case with HeLa and Wi—38, this o-polymerase was also sensitive.

Recently, Miller and Rapp (33) reported that the o-polymerase of HEL,

50
a cell line with a finite life expectancy originating from normal
human embryonic lung tissue, was also sensitive. Thus, it is clear,
when all of these results are taken together, that the human
a-polymerase is sensitive to phosphonoacetate.

Other vertebrate eukaryotic o-polymerases were also observed to
be sensitive to phosphonoacetate. Calf thymus and CHO o-polymerase
showed inhibition. Leinbach et a1. (9) have reported that the
o-polymerase from duck embryo fibroblasts is also inhibited by
phosphonoacetate.

The B-polymerase of three human cells, HeLa, Wi-38 and lympho-
cytes, is relatively insensitive to phosphonoacetate. The result
for HeLa is in agreement with that of Bolden et al. (12) and the
result for Wi-38 agrees with those reported by others (2,6,7,ll).
Moreover, the B-polymerase of CHO cells, as reported here, and the
B-polymerase of duck embryo fibroblasts as previously reported by
Leinbach et al. (9) are not inhibited by phosphonoacetate. As
observed by Knopf et al. (29) with HeLa y-polymerase, the y-polymerase
of fetal calf liver was also relatively insensitive to phosphono-
acetate. Therefore, the sensitivity of the o-polymerase and the
relative insensitivity of the B- and y-polymerases appears to be a
general characteristic of vertebrate polymerases.

Yeast, a simple eukaryote, contains two DNA polymerases in
mitochondrial free cell extracts, designated A and B, rather than
the three DNA polymerases, a, B, and y, of vertebrate eukaryotes
(24). In this report, both of the yeast polymerases were observed
to be sensitive to phosphonoacetate and to about the same extent as

o-polymerase. A single DNA polymerase in tobacco cells (unpublished

51
work) was identified. In this report, the DNA polymerase from
tobacco cells was also shown to be sensitive to phosphonoacetate.

Of the many phosphonate compounds which were examined for
inhibition of the HeLa o-polymerase, only one, phosphonoformate, was
shown to be an inhibitor. Phosphonoformate was also an inhibitor of
the yeast DNA polymerase A and B and the tobacco cell DNA polymerase.
Recently, in this laboratory, Reno et al. (submitted for publication)
discovered that phosphonoformate is an effective inhibitor of
herpesvirus-induced DNA polymerase and that the mechanism of inhibi-
tion by phosphonoformate is analogous to that of phosphonoacetate.
Leinbach et al. (9) reported that phosphonoacetate inhibits the
herpesvirus-induced DNA polymerase by interacting at the pyrophosphate-
binding site. For the experiments reported here with HeLa o-polymerase,
phosphonoformate and phosphonoacetate were shown to be mutually
exclusive inhibitors which bind at the same site on the a-polymerase.
Presumably, this site is the pyrophosphate-binding site.

At high concentrations, about ten to twenty times greater than
that which effectively blocks herpesvirus replication, phosphono-
acetate is cytotoxic (2,5,10) and cellular DNA synthesis is inhibited.
Although not proven, it is not unreasonable to assume that at high
concentrations, phosphonoacetate inhibits cell growth as a result of
the inhibition of cellular DNA synthesis through a specific inhibi-
tion of the o-polymerase. This phosphonoacetate inhibition of
cellular DNA synthesis by inhibition of the o-polymerase would be
analogous to the inhibition of herpesvirus DNA synthesis by inhibition
of the herpesvirus-induced DNA polymerase (7). The difference would
be in the concentrations of phosphonoacetate required to inhibit the

two processes. If this is so, then phosphonoacetate-resistant cell

52
mutants could contain an altered o-polymerase. These mutants might
prove useful in evaluating the role that a-polymerase plays in

cellular DNA replication.

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