Eukaryotic DNA polymerases.

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Annu. Rev. Biochem. 1977.46:25-47. Downloaded from www.annualreviews.org Access provided by University of California - San Francisco UCSF on 01/28/15. For personal use only.

Ann. Rev. Biochem. 1977. 46:25-47 Copyright © 1977 by Annual Reviews Inc. All rights reserved

EUKARYOTIC DNA

+938

POLYMERASES Arthur Weissbach Department of Cell Biology, Roche Institute of Molecular Biology, Nutley, New Jersey 07110

CONTENTS PERSPECTIVES A ND SU MMARY.. .. ... ....... .......... .................. ...... .. . .. . .. .. .. .. .

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IN TR ODUCTION .. . ...... .... ...... .. .. ... . . .. ... . .. .. .. .. ........ .. ..... .. ...... .... .... .. .. .. .

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CE LLU LAR DNA POLYMERASES .. .. ..... . . ............ ..... .. .. .. ...... ...... ... . ...... .

DNA Polymerase

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Mitochondrial DNA Polymerase.. . . . .. ....... .. .. .. .. .. .. .. .. ...... .. . . . .. . ....

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a

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DNA Polymerase /3 . . .... . .. . .. ..... ... .... .. .. . .

DNA Polymerase 'Y

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VIR US-INDUCED DNA POLYMERASES .. .. .. ..... . ... .. ....... .. .... .. . . .. ....... .. ...

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DNA Polymerases Induced by Herpesvirus ........ .. ........ ..... .... ......... .... .. .. . DNA Polymerase Activities of Parvoviruses and the Hepatitis B Particle..

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Changes Induced by Papovavirus and Adenovirus Infection

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COMPARA TIVE STUDIES OF THE DNA POLYMERASES................................

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Lack of Interrelationship of the Enzymes .. . ..... .. .. .. ..... .. ... .. . .. . ..... .. ..

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DNA Polymerase Activities and DNA Synthesis in Vivo................................ DNA Polymerases during Development.............................................................. Template Studies . . .... .. ...... .. .. .. .. .. .. .... ........... .. ... .... .. .. .. . .. . .... .. .. . .....

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Other DNA Polymerases........................................................................................

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CONCLUSION................................................................................................................

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PERSPECTIVES AND SUMMARY Higher eukaryotic cells contain at least three distinct DNA polymerases, which have been named DNA polymerases

a,

/3, and 'Y. These DNA

polymerases can easily be distinguished from one another by their chromatographic properties, molecular weight, sensitivity to N-ethylmalei25


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mide and salts, and ability to copy various templates.1 Mitochondria, as isolated from mammalian cells, also contain a DNA polymerase activity that has been considered to be different from the other cellular DNA polymerases. However, recent evidence has suggested that the mito chondria-associated DNA polymerase may be a form of DNA polymer ase y. The apparent location of the DNA polymerases in the cell has raised some questions about their possible roles. When cells are fractionated into nuclear and cytoplasmic components in aqueous solutions, the a-polyme rase, which is often the major activity in growing cells, frequently appears in large amounts in the cytoplasm. However, this may be an artifact since other techniques, which involve nonaqueous breakage of cells or drug induced enucleation, indicate that 80-90% of the total DNA polymerase activity is, in fact, associated with the nucleus. Because almost all of the cellular DNA synthesis normally occurs in the nucleus, localization of the DNA polymerases there is to be expected. Infection of animal cells with viruses often leads either to a change in the cellular DNA polymerase levels or to the appearance of a new virus induced, and presumably virus-encoded, DNA polymerase. Polyoma virus or SV40 infection of cells causes a five- to tenfold rise in the cellular levels of DNA polymerases a and y and a doubling of the i3-polymerase activity. Viruses containing large DNA genomes, such as those in the herpes group or pox group, produce a new DNA polymerase in infected cells. Genetic studies with herpes simplex virus have indicated that the virus-encoded DNA polymerase is required for viral DNA synthesis. Two small DNA containing viruses, the hepatitis B Dane particle and the rat Kilham virus, have also been found to contain a DNA polymerase activity within the virus particle itself, although the identity of the virus-associated enzyme is un known. The structure of the eukaryotic DNA polymerases remains unresolved, although it has been shown that both the a- and y-polymerases have molecular weights of over 100,000 and thus differ from the i3-polymerase, which has a molecular weight of 30,000-50,000. Immunological and bio chemical studies suggest that the three cellular DNA polymerases are not related to one another, do not share common peptide sequences, and are not interconvertible. However, the DNA polymerase a of HeLa cells seems to share some similarities with the a-polymerase of hamster cells, and a corre sponding cross-species relationship has been noted with the avian and mamlIn this text "template" is defined as the polynucleotide strand that is copied by a DNA polymerase and "primer" as the polynucleotide strand containing a 3'-OH end group from which the growing DNA chain is covalently extended.


EUKARYOTIC DNA POLYMERASES

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malian ,8-polymerases. Phylogenetic studies have also indicated that the a- and IJ-polymerases are widely distributed among eukaryotes, and a form of DNA polymerase 'Y has been found in many animal cells and even in wheat germ. The function of the DNA polymerases in the complex process of DNA replication is not known. Correlative studies of DNA synthesis and the level of DNA polymerase activity suggest that DNA polymerase a may be important in DNA replication. However, in certain cases, DNA polyme rases ,8 and 'Y also increase at the time of DNA synthesis, and attempts to specify which of the DNA polymerases is "replicative" or to assign roles to the various polymerases have not yet been successful. It is extraordinary that both prokaryotes such as Escherichia coli and higher cells contain, at the present accounting, three major DNA polymerases; understanding the reason for this multiplicity will be an essential part of deciphering the process of DNA replication. It is also clear that the functioning of the DNA polymerases may be affected by or dependent upon the other chromatin and nuclear proteins constituting the DNA replication complex and that the enzymes may have to be studied within that context.

INTRODUCTION During the past few years considerable information has accumulated con cerning the type and number of DNA polymerases in eukaryotic cells. An international conference on eukaryotic DNA polymerases held in 1975 at the Asilomar Conference Center, Pacific Grove, California, led to a formal adoption of a nomenclature to describe these enzymes (1, 2). This nomen clature is shown in Table 1 and is intended to help organize the large number of previous studies on DNA polymerases and provide a framework for future studies. The cellular DNA polymerases have been assigned Greek letters in order of their historical discovery (Table 1), with the exception of the DNA polymerase activity found in mitochondria, which has been assigned a separate name to mark its location. It should be emphasized that the number, type, and relationship of the enzymes that are presently known may be expanded and modified in the near future. Furthermore, the bulk of data concerning eukaryotic DNA polymerases has been garnered from studies with vertebrate cells, and an interspecies comparison of the DNA polymerases within the animal kingdom as well as those in plants has just begun. This phylogenetic search for the DNA polymerases should be of great value and may give a clue to their function. In addition to the cellular enzymes shown in Table 1 there are, as shown in Table 2, a number of virus-induced DNA polymerases, which appear in cells after viral infection and which are named after the viruses that induce, and presumably code

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Table 1 Nomenclature for the eukaryotic cell DNA polymerases

Molecular weight

Inhibition by N-ethylmaleimide

120,000-300,000

+


DNA polymerase

30,000-5°,000

Salt effect Inhibited at NaCI concentrations above 25 mM Stimulated by 100200 mM NaCI but in hibited by 50 mM

POr 'Y

150,000-300,000

+

Stimulated by 100250 mM KCl and 50 mM

POr Mitochondrial (mt)

150,000

+

Stimulated by 100200 mM KCl

for, these new enzymes. The Type C viral reverse transcriptases are the most extensively studied enzymes in this class and have been the subject of several recent reviews (3-5). Neither reverse transcriptase nor terminal deoxynu cleotidyl transferase is considered in this review. The latter enzyme is considered to be a nonreplicative enzyme of unknown specialized function and has been discussed previously (6). The present review, extending to July 1976, deals with the mammalian DNA polymerases and those induced by the DNA-containing viruses that infect these cells. The reader is referred to other reviews in this area that serve as a background for this discussion (7-9).

CELLULAR DNA POLYMERASES DNA Polymerase

a

DNA polymerase a was the first of the mammalian DNA polymerases to be partially purified and studied (10). It seems to be ubiquitous in growing cells and has been found in calf thymus (11-13), human (14), murine (15), and hamster cells (16, 17), and in avian tissues (18), sea urchins (19, 20), and yeast (21) among others. A characteristic property of the a-polymerase is its sensitivity to sultby dryl group blockers such as p-hydroxymercuribenzoate and N-ethylmalei mide (2, 7, 22). The enzyme is also inhibited by salt concentrations above 25 mM (10) and has an absolute requirement for a divalent cation such as MgH or MnH. Neither the human enzyme nor the calf thymus enzyme


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Table 2 Virus-induced DNA polymerases A)

DNA-Containing Viruses

1)

Herpes Group Herpes simplex virus Marek's disease herpesvirus Herpesvirus of turkeys


Cytomegalovirus Pseudorabies Equine herpesvirus

2) Pox Group Vaccinia 3)

Viruses Containing DNA Polymerases Kilham rat virus Hepatitis B Dane particle

B)

RNA-Containing Viruses

C)

Induction of Host DNA Polymerases

Type C oncornavirus (reverse transcriptase)

Polyoma SV40

shows associated nuclease activities (7, 14). The calf thymus a-polymerase has been reported to catalyze pyrophosphate exchange (7). Two of the liveliest areas of research with DNA polymerase a involve the size and composition of the enzyme and its intracellular location. Molecular weight determinations or determinations of sedimentation velocity are widely used and, as discussed later, often deceptive criteria for recognition of the DNA polymerase. In the case of the a-polymerase, molecular weights from about 70,000 to over 1,000,000, with S values of 5-12, have been reported in several species. The purified human enzyme, having a specific activity of 7,300 units/mg protein, has been reported to show a native molecular weight of 175,000 and to contain an 87,000-dalton peptide subunit. The enzyme may also exist in aggregated forms in solutions of low ionic strength, with molecular weights in the range of 300,000 (7, 14, 23). DNA polymerase a of calf thymus, which has a specific activity of at least 15,000 units/mg, is a single polypeptide of molecular weight 155,000-170,000 and seems to contain an additional subunit of 50,00070,000 daltons (7, 12). Brun et al (18) found the bulk of the chick embryo a-polymerase to have a molecular weight of 148,000, but also found higher molecular-weight forms. Craig & Keir (17) have investigated the DNA polymerases of BHK-211CI3 cells and tentatively identified four subspecies of the a-polymerase ranging in apparent molecular weights from 140,000


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to 1,000,000. They have noted, as did Holmes et al previously (24), the molecular asymmetry of DNA polymerase a and lack of correlation of molecular weights as derived from gel filtration or sedimentation coeffi cients. Since the molecular heterogeneity of the a- polymerase appears to be a general phenomenon (7, 15, 25), it is not always possible to assign a simple relationship between the various molecular-weight and chromato graphic forms of the a-polymerase and their enzymatic activity. In this respect Mechali & De Recondo (26) have found an endonuclease in rat liver that purifies together with DNA polymerase a and introduces single-strand nicks in DNA. Although it can be separated from the a-polymerase by affinity chromatography on DNA cellulose, the authors suggest that the endonuclease may physically interact with the a-polymerase and thus have a potential role in DNA replication. The possibility that the a-polymerase may form complexes with acces sory proteins such as the nucleic acid helix-unwinding proteins from calf thymus has also been raised by Herrick et al (27). The calf thymus unwind ing protein UPI will stimulate the activity of DNA polymerase a but not that of DNA polymerase f3 or 'Y (27, 28). Phage T4 gene-32 protein will not replace the calf thymus protein. Baril & Baril (29) have reported that DNA polymerase a can be found with membrane fragments containing ribonucleotide reductase, thymidylate synthetase, and thymidine kinase. If these putative, or analogous, multienzyme complexes could be shown to participate in DNA replication, they would provide an important tool for further studies. The role of cations in DNA synthesis must also be consid ered, since Yoshida et al (30) have found that polyamines affect in vitro DNA synthesis carried out by the calf thymus DNA polymerases. In the past, DNA polymerase a has been referred to as the "cytoplasmic" DNA polymerase because the cytoplasm obtained from cells broken by common aqueous procedures contains large amounts of the enzyme. Al though this is a useful operational tool, it is probable that the in vivo intracellular location of the enzyme is in the nucleus. Using cytochalasin induced enucleation,Herrick & co-workers have demonstrated that greater than 85% of the DNA polymerase activity is associated with the nucleus (31). In addition, Siebert et al (32) have obtained similar results using nonaqueous techniques with organic solvents to isolate nuclei. Foster & Gurney have prepared mouse fibroblast nuclei in glycerol and found only 10% of the cellular DNA polymerase activity to be outside the nucleus (33). Finally, Lynch et al (34) have isolated hepatic nuclei in aqueous solution of low ionic strength or in glycerol and found them to contain nearly all of the nonmitochondrial DNA polymerase of the cell. The conclusion from these studies is that the major part of DNA polymerase a and f3 activity is located in the nucleus of animal cells. Nevertheless; it is not clear that



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a-polymerase is located only in the nucleus since it has been reported that leukemic cells have a nuclear a-polymerase that differs somewhat from that in the cytoplasm (35). It is well known that DNA polymerase a is very active with DNA primer-template combinations, which are formed by the action of DNase I on duplex DNA (activated DNA). The ability of calf thymus DNA polymerase a to utilize DNA templates has been reexamined by Wang et al (36). As expected, double-stranded and single-stranded DNA's are inac tive as templates unless they are activated by nuclease treatment to produce sufficient 3'-OH ends. The product of the reaction when DNA polymerase a acts on nuclease-treated DNA was found to be double-stranded DNA with hairpin structures. The a-polymerase is rather specific for polydeox yribonucleotide . templates and shows little activity with synthetic ribohomopolymers such as poly(A) or RNA. It thus resembles E coli DNA polymerase I. Like the E coli enzyme the a-polymerase can also catalyze the de novo synthesis of the copolym:er poly [d(A-T)od(A-T)] (37). The reaction, which has a lag period of 1-2 hr, occurs in the presence of the calf thymus unwinding protein UPI and in the presence of dATP and dTTP.

DNA Polymerase /3 This low-molecular-weight DNA polymerase was first identified in HeLa cell and rat liver nuclei in 1971 (22, 38), and shortly thereafter in calf thymus (39), and then further characterized in rat liver (40, 41). The enzyme has now been purified to homogeneity from calf thymus (42) and human (KB) cells (43) and to near homogeneity from chick embryos (18). The latter enzyme is probably identical to that reported by Stavrianopoulos et al (44). The purified enzyme from calf tQY�s or KB cells or rat ascites cells (45) has a molecular weight of about 45,000, shows a sedimentation value of 3-4S, and has a specific activity of 200,000 units/mg of protein (calf thymus) or 8,000-9,000 units/mg (KB cells). The avian /3-polymerase ex ists as a 27, OOO-dalton form in the cytoplasm and a 50, OOO-dalton dimeric form in the nucleus. The segregation of the dimer to the nucleus may indicate an active role of this form in DNA synthesis and a specific nuclear mechanism for monomer to dimer interconversion (18). Probst et al have recently reported on several proteins from Novikoff hepatoma that markedly stimulate the isolated Novikoff /3-polymerase (46). Whether these accessory proteins interact directly with the /3-polymerase to form an active complex remains to be clarified. The ,a-polymerase has no requirements for sulfhydryl groups and is not markedly inhibited by low levels (0.025mM) of p-chloromercuribenzoate, which would completely inhibit the a-polymerase (22). The differential sensitivity of the ,a-polymerase to p-chloromercuribenzoate, N-ethylmalei-


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WEISSBACH

mide, and other sulfhydryl blocking agents are fully discussed by Matsuk age et al (47). Concentrations of p-chloromercuribenzoate or HgCl2 above 0.1 mM will, however, markedly inhibit the fi-polymerase, whereas little inhibition of the enzyme is seen with N-ethylmaleimide at concentrations of up to 3 mM. The fi-polymerase is, in general, an enzyme showing a high resistance to a number of chemical agents. It is not inhibited by phos phonoacetic acid (48), 5 M urea, or 20-25% ethanol or acetone (42) and is stable in solution at pH 4.5 to 10.5. The purified enzyme is free of nuclease activity; it does not carry out pyrophosphate exchange (42, 43). It has been repeatedly noted that the fi-polymerase, in the presence of an activated DNA template, shows a significant ability to incorporate a single deoxynucleoside triphosphate into an acid-insoluble form. This is due not to contamination with terminal deoxynucleotidyl transferase but to a limited repair synthesis occurring at the large number of 3'-OH ends in the template. Thus significant incorporation is not seen when a full duplexed molecule such as poly[d(A-T).d(T-A)] is used (49-51). Chang & Bollum (7, 52) have examined the primer (initiator) and template specificities of the a- and fi-polymerase in some detail using synthetic polydeoxynucleotide templates and complementary oligoribo- or oligodeoxyribonucleotide prim ers. It is clear that the /3-polymerase, but not the a-polymerase, can copy a poly(A) template, although it is apparently unable to utilize poly(C) as a template, in the presence of the appropriate oligodeoxynucleotide primer. In general, however, the fi-polymerase utilizes polydeoxyribonucleotide templates such as poly(dA) or poly(dC) more efficiently than the corre sponding polyribonucleotides, poly(A) or poly(C). By criteria mentioned in the discussion of the a-polymerase, the /3polymerase is considered to be located solely in the nucleus (22, 31-34), although breakage of cells can lead to introduction of the enzyme into the cytoplasm (7,53). Release of both the a- and fi-polymerases from isolated nuclei by ATP has also been reported (54). The a- and fi-polymerases are also found in the nucleoli isolated from Ehrlich's ascites tumor cells (55). Earlier studies indicated that DNA polymerase fi was probably an ubiquitous enzyme in the animal kingdom. A valuable study on the phy logeny of DNA polymerase fi has now been carried out by Chang (56), who showed that this enzyme class is widely distributed in multicellular animals but is absent in bacteria, plants, and protozoa. Interestingly, Chang also looked at a-polymerase activity in her survey and found this class of DNA polymerase to be present in all eukaryotes examined. The enzymes classified in this study were identified by their molecular weight and sensitivity to N-ethylmaleimide; further examination and characterization of these DNA polymerases would be useful. This study is important in that it suggests new avenues for approaching the significance of the eukaryotic DNA polyme rases.


33


DNA Polymerase y DNA polymerase "I is a widely distributed enzyme (8) that can copy, in addition to natural or synthetic DNA templates, a variety of synthetic ribohomopolymers such as poly(A), poly(C), etc (57-59). Compared with the �-polymerase, DNA polymerase "I prefers a ribohomopolymer as a template and will copy, for instance, poly(A) 5-10 times faster than poly(dA). This preference for synthetic ribohomopolymers rather than nat ural or synthetic DNA templates is a characteristic property of "I-polyme rases including those isolated from such varied sources as human tissues (57-61) and wheat embryos (62). The "I-polymerase has not yet been puri fied to homogeneity, but preparations having a specific activity of 25,000 units/mg of protein after a 60,000-fold purification have been obtained from HeLa cells (58). Like the a- and �-polymerases, the "I-polymerase shows a heterogeneous nature when isolated, and molecular weights from 110,000 to 300,000 have been reported for the human enzyme (57, 58). Matsukage and co-workers have studied a murine "I-polymerase and reported molecu lar weights of 230,000-315,000 for various forms of the native enzyme (63). Multiple forms of the "I-polymerase as isolated from calf thymus have also been noted by Yoshida et al (64). Because of the propensity of the "1polymerase to form aggregates, particularly under conditions of low ionic strength (63), it has been difficult to assess the significance of the diverse forms of this enzyme that have been reported. Purification and isolation of the "I-polymerase in a homogeneous form is needed so that the structure of this enzyme can be compared with that of the other cellular polymerases and with the RNA-dependent DNA polyme rases of Type C RNA tumor virus. Both the "I-polymerases and viral reverse transcriptases show the same efficient use of ribohomopolymer templates such as poly(A), although the "I-polymerase has not been demonstrated to copy a natural RNA template despite many attempts in this direction. Immunological studies have shown that blocking antiserum prepared against the reverse transcriptases of Rauscher leukemia virus, Woolly mon key virus, Mason-Pfizer virus, or the feline virus RD-114 will not inhibit the human "I-polymerase (57, 65). In addition, the cellular "I-polymerase and virus-induced reverse transcriptases can be clearly separated and identi fied in Type C virus-infected cells (61, 66). A further distinction between the 'Y-polymerase and reverse transcriptases has been reported by Gerard (59), who found that a poly (2'-O-methylcytidylate) template cannot be copied by HeLa cell or murine (3T6 cells) DNA polymerase "I, whereas it is an effective template for every RNA tumor virus RNA-dependent DNA polymerase so tested (59, 67). It should be noted, however, that Mizutani & Temin (68) have found specific serological relationships among the par tially purified DNA polymerases of avian cells and the RNA-dependent


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WEISSBACH

DNA polymerases of avian Type C tumor viruses. This type of affinity between these reverse transcriptases and the cell enzymes is, so far, unique and has only been reported in the avian system. The'Y-polymerase can be easily distinguished from the a- and ,8-polyme rases by several criteria. The activity of human DNA polymerase 'Y is optimal in 100-300 mM KCI and the enzyme is stimulated by phosphate in the presence of KCl. As mentioned previously a-polymerase activity is markedly inhibited at salt concentrations above 25 mM (10) and ,8-polyme rase activity is also considerably diminished in the presence of phosphate (49,58). Like the a-polymerase, and in contrast to the ,8-polymerase, DNA polymerase'Y is also inhibited by N-ethylmaleimide (50-80% at 1 mM) (58, 63). These characteristics, coupled with the known molecular weights, enable one to distinguish between each of the three known cellular DNA polymerases in human cells.

Mitochondrial (mt) DNA Polymerase From the first studies of Kalf & Ch'ih (69) and Meyer & Simpson (70, 71) it has been assumed that mitochondria, and perhaps other cellular or ganelles, possess a unique DNA polymerase that differs from the other

DNA polymerases. DNA polymerase mt is reported to be a salt stimulated enzyme with a molecular weight of about 150,000 as isolated from rat or mouse liver (72, 73). The enzyme from yeast mitochondria (74), or as obtained in one study from HeLa cells (75), also has a molecular weight of about 150,000 but is not stimulated by the addition of salt. The enzyme has not been highly purified from these sources but has been re ported to use single-stranded, double-stranded, and closed circular duplex DNA as templates, though less well than activated DNA. Since single stranded or fully duplexed DNA is, in general, a poor template for purified DNA polymerases unless significant nuclease activity is present to generate 3'-OH priming ends, further purification of the mitochondrial DNA polymerase from these sources is needed to understand these results. The enzyme has also been reported to be inhibited by ethidium bromide and ATP (73). Another study with mitochondria isolated from HeLa cells claimed that two DNA polymerase activities were present (76). One of these was DNA polymerase'Y and the other a new DNA polymerase, which has a molecular weight of about 106,000 and was quite different from the other cellular enzymes. This latter activity also found in preparations of mitochondria of KB cells (77) and has recently been further studied in HeLa cell mito chondria (78) purified by repeated centrifugation. This 106,OOO-dalton en zyme was considered to be the mitochondrial DNA polymerase (78). However, recent work has shown that these cultural HeLa cells were concellular



35

taminated with mycoplasma and the 106,OOO-dalton enzyme was probably a mycoplasma-derived contaminant. Mitochondria from mycoplasma-free HeLa cells or from rat liver contain only one DNA polymerase activity, which is very similar to the corresponding cellular 'Y-polymerase (A. Bolden, G. Pedrali Noy, A. Weissbach, 1977, 1. Bioi. Chem., in press). If confirmed, these latter studies would indicate that there are three, rather than four, cellular DNA polymerases in higher cells and that there is no uniquely different class of mitochondrial DNA polymerases.

VIRUS-INDUCED DNA POLYMERASES The DNA polymerases induced in cells after infection with vaccinia virus, herpes simplex virus (HSV), or Marek's disease herpesvirus were partially reviewed in 1975 (8).

DNA Polymerases Induced by Herpesvirus The HSV-induced DNA polymerase has received considerable attention recently as research on this virus increases. First discovered and identified by Keir et al (79, 80), the enzyme has been further studied in infected HeLa cells (81) and rabbit kidney cells (82). The HSV-induced DNA polymerase has a high molecular weight (180,000) and is maximally active in 0.15 M K2S04 or 250 mM KCl (81,83), which are salt concentrations that strongly inhibit the a-polymerase. Under assay conditions using high salt concentra tions, the virus-induced DNA polymerase is, therefore, easily detected in infected cell extracts or cytoplasmic fractions. Studies with DNA-negative, temperature-sensitive mutants of HSV- l or HSV-2 have indicated that the HSV-induced DNA polymerase is encoded in the viral DNA and that it is necessary for viral DNA synthesis. Synthesis of the enzyme controlled by HSV-l requires at least two viral genes (84) and formation of the HSV-2 DNA polymerase involves at least three herpes genes (85). There is now increasing evidence that other members of the herpes group of viruses are capable of coding for new DNA polymerases. As a class, these are viruses that contain relatively large DNA genomes, ranging from 60 to 100 X 106 daltons, and therefore have sizable coding capacities. Thus a productive human cytomegalovirus infection of human cells produces a novel DNA polymerase activity that, like the HSV-induced DNA polyme rase, is activated by high salt concentrations (86). By contrast, nonpermis sive guinea pig cells, infected by cytomegalovirus, show an increase in DNA polymerase activity that is not salt-dependent and probably represents an increase of the host polymerases. Pseudorabies infection of BHK cells also leads to the appearance of a DNA polymerase that is stimulated by high


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WEISS BACH

salt concentrations and is inhibited by antiserum to pseudorabies-induced proteins (87). In addition, the formation of a high-salt-dependent DNA polymerase has been note:d between 2 and 9 hr after infection of hamster hepatic cells with equine herpesvirus Type 1 (88). There have been reports of a new enzyme in cells infected with Marek's disease herpesvirus or the herpesvirus of turkeys, which has a molecular weight of 100,000 and which is inhibited by high salt concentrations (89). Recent work has indicated that this enzyme is not a virus-induced product and that these infected cells also contain a high-salt-stimulated DNA polymerase that is presumably virus directed (1. Boezi, personal communication). The demonstration that the HSV-induced DNA polymerase is required for viral DNA replication has led to a search for antiviral compounds that might selectively block this enzyme. A group at Abbott Laboratories has reported that phosphonoacetate is a specific inhibitor of the HSV-induced DNA polymerase (90, 91). Phosphonoacetate is also an inhibitor of the DNA polymerases induced by the herpesvirus of turkeys (92) and human cytomegalovirus (93). However, Bolden et al (48) reported that phosphon oacetate is an inhibitor of the vaccinia virus-induced DNA polymerase and

HeLa cell DNA polymerase a. The compound showed a moderate inhibi tory action against DNA polymerase 'Y and no effect on the /3-polymerase. Since phosphonoacetate is an inhibitor of the virus-induced DNA polyme rases mentioned above, it is of some interest that Epstein-Barr virus forma tion is also inhibited by this drug (94-96). This might indicate that this herpesvirus, which is associated with mononucleosis and Burkitt's lym phoma, may induce a new virus-directed DNA polymerase.

DNA Polymerase Activities of Parvoviruses and the Hepatitis B Particle As far as is known, the virus-induced DNA polymerases formed in herpes

or vaccinia virus infections are localized in the cells and are not found in the virion as is the case with the oncornavirus reverse transcriptase. Inter estingly enough, two viruses containing a relatively small DNA genome, Kilham rat parvovirus (97) and the hepatitis B Dane particle (98), have been reported to carry a DNA polymerase activity within the virion. In both cases it is not clear if these associated enzyme activities represent new, virus-induced enzymes or host enzymes that become associated with the virus. In view of the small size of the genome of these viruses, the latter possibility seems more likely. However, Salzman & McKerlie (97, 99) have compared the parvovirus-associated DNA polymerase with the DNA polymerases found in the rat nephroma host cells. The DNA polymerase associated with Kilham rat virus shares some properties with the host DNA polymerases yet possesses some unique characteristics, so that the issue



37

remains unresolved. The identity of the DNA polymerase associated with the hepatitis B antigen is even more obscure. The enzyme and its template are intimately associated with the Dane particle, which is the presumed hepatitis B virion, and have resisted solubilization. Even the DNA product synthesized in the Dane particle is tightly attached to the core and not susceptible to attack by deoxyribonucleases ( l 00). Because of the apparent in situ restraints, the Dane particle-associated DNA polymerase does not respond to exogenous templates. When and if this bound enzyme is freed from the virion, it should be of interest to compare it to the known human DNA polymerases.

Changes Induced by Papovavirus and Adenovirus Infection Two oncogenic DNA viruses, polyoma and SV40, are known to induce cellular DNA synthesis upon infection and cause a rise in DNA polymerase activity. It has now been established that both DNA polymerases a and Y increase five-to tenfold in the nuclei after polyoma infection of 3T3 cells, whereas the l3-polymerase activity doubles (101). Essentially the same in crease of a-polymerase activity was noted in polyoma-infected resting mouse kidney cells by Wintersberger & Wintersberger (102), although they noted no increase in the l3-polymerase level. The increase in DNA polyme rase activity seen in infected cells is thus similar to the pattern generally seen when quiescent cells are stimulated to divide. An increase in the DNA polymerase activity of cultured cells infected with African swine fever virus (an iridovirus) has also been reported (103). Ito et al (104) have isolated a nuclear membrane complex from KB cells infected with adenovirus 2 that has some properties of an adeno DNA replication complex. The major DNA polymerase in this complex, which can synthesize viral DNA, is DNA polymerase y. It remains to be deter mined if, in fact, the y-polymerase plays a major role in adenovirus DNA synthesis-this would represent the first known role of a specific host DNA polymerase in the DNA replication of an animal virus.

COMPARATIVE STUDIES OF THE DNA POLYMERASES Lack of Interrelationship of the Enzymes The identification of three or four cellular DNA polymerases leads one to question if these enzymes are interrelated or share any common polypeptide sequences. Early work with antiserum to calf thymus DNA polymerase a (105) and sedimentation studies purporting to show an interconversion between the a- and l3-polymerases (106-108) suggested a possible relation ship between these enzymes. Recent studies (109-111) have indicated the


38

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opposite, i.e. that there is no structural relationship or polypeptide sharing between DNA polymerases a, /3, or 'Y. Three groups of researchers have prepared antiserum to either human DNA polymerase a (109,110) or avian DNA polymerases a and {3 (111). The antiserum to the human a-polyme rase does not inhibit the activity of the human {3- or 'Y-polymerases or the RNA-dependent DNA polymerases from avian myeloblastosis virus, Rauscher leukemia virus, Mason-Pfizer virus, simian sarcoma virus, and gibbon ape lymphosarcoma virus. Conversely the avian /3-polymerase an tiserum will not inhibit the avian a-polymerase or the avian myeloblastosis reverse transcriptases. The work with antiserum to human DNA polyme rase a indicated that it would cross-react with hamster cell DNA polyme rase a (109). A similar phenomenon has been seen with the avian /3polymerase antiserum, which also cross-reacts with mammalian /3-polyme rases (111), although the antiserum to the avian a-polymerase did not seem to inhibit hamster cell a-polymerase. The comparison of immunological relatedness needs much more study, but the data suggest that the a-polyme rases from many species may share common polypeptide sequences, and one can speculate that there may be partial homology of the /3-polymerases found throughout the metazoa (56). As discussed previously in this review, immunologic studies have also indicated that there is no apparent structural relationship between any of the known oncornavirus reverse transcriptases and DNA polymerases a, /3, and 'Y. Bolden et al (48) have also tested antiserum prepared against HSV- or vaccinia virus-infected cells and have shown no immunologic cross-reactivity between the host and viral DNA polymerases examined. Thus, antiserum to HSV-induced proteins will specifically inhibit the HSV DNA polymerase to over 90% but not the vaccinia DNA polymerase (or HeLa cell DNA polymerase a, /3, or 'Y; J. Aucker, A. Weissbach, unpub lished). Similarly, antiserum to the vaccinia-induced polymerase, which inhibits this enzyme up to 85%, will not affect the activity of the herpes DNA polymerase or any of the cellular DNA polymerases. Finally, an tiserum to HeLa cell DNA polymerase a (109) will not inhibit the herpes or vaccinia-induced DNA polymerase (43). These data coupled with the known biochemical and physical differences of the enzymes suggest that the viral and host DNA polymerases share little, if any, common peptide sequences. The final proof of this hypothesis awaits preparation of rigor ously pure DNA polymerases and knowledge of the amino acid sequence. Lacking this data, it is not possible to completely exclude the possibility that the virus-induced DNA polymerases represent modifications of host en zymes or contain fragments thereof. As mentioned above, there have been studies of cellular DNA polyme rases that used sedimentation velocity criteria to distinguish the a- and {3-



39

polymerases and suggested that the a- and ,8-polymerases might be physi cally interconvertible (106, 107). The interpretation of these results has been questioned subsequent to the demonstration that DNA polymerase ,8 can form aggregates in vitro (43, 112). These aggregate forms can show sedi mentation values of 6-8S and hence can be confused with the a-polymerase, which shows similar sedimentation characteristics. At the present time, therefore, no evidence exists for any relationship between the a- and ,8polymerases. However, there may be subclas�s within these groups, and one should recall, as mentioned previously, that each of the cellular DNA polymerases sometimes seems to exhibit heterogeneity during isolation pro cedures. This may indicate. that a given enzyme, for example the a-polyme rase, may exist in several forms containing modifications or may perhaps be associated JVith accessory proteins. Reisher et al (113) have found that calf thymus DNA polymerase a can be phosphorylated by a cyclic AMP dependent protein kinase and that phosphorylation appears to stimulate the DNA polymerase reaction. In addition, evidence has been given that pro tein phosphorylation and dephosphorylation may regulate the activity of the reverse transcriptase of Rous sarcoma virus (114).

DNA Polymerase Activities and DNA Synthesis in Vivo The steps by which mammalian DNA is synthesized are unclear, but it is generally assumed that DNA replication occurs in discontinuous units arranged in a tandem manner on the chromosome (115-117). The earliest intermediates so far detected in the DNA replication process are apparently oligodeoxynucleotides of relatively short sizes, below 200 nucleotides in length (118, 119), which then eventually are joined to form high-molecular weight DNA. In addition, evidence has been presented that initiation of DNA synthesis may involve growth of the DNA chain from an RNA primer (120-122). With this evidence that there are discrete stages in the formation of the DNA strand, a number of investigations have focused on the possible role of each of the DNA polymerases in this complex and unknown process. Many of the studies have been correlative and concerned with measuring the activity of each of the DNA polymerases in cells under varying condi tions of growth or quiescence. Chang & Bollum (123) found that when L cells are stimulated to divide there is a rise in the a-polymerase activity with little change in the level of the ,8-polymerase. Investigations with regenerat ing rat liver 18-30 hr posthepatectomy also showed similar results (1�4), and induced erythropoiesis in mouse spleen again leads to a rise in the a-polymerase levels, but not the ,8-polymerase (125, 126). It is a general observation that growing cells contain a high level of DNA polymerase a that may represent as much as 90% of the total cellular DNA polymerase


40

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activity. In quiescent BHK. tissue culture cells or cells in the Go state, the p-polymerase is the predominant activity (127). A rise in the a-polymerase level can also be seen in experimental neoplasia of rat liver, which is induced by a hepatocarcinogenic diet (128). Some of these studies have noted changes in the relative distribution of DNA polymerase activity between the nucleus and cytoplasm in cells undergoing growth or in cells entering the DNA synthetic phase (S phase) of the cell cycle. Evidence presented earlier in this review (31-34) indicates that certain cell fractionation procedures may lead to an artifactual distribution of DNA polymerase activity between nuclei and cytoplasm; consequently, it is difficult to evaluate such data. Investigations with synchronized tissue culture cells have shown a posi tive correlation between the rate of DNA synthesis and the activity of the a-polymerase. Spadari & Weissbach (129) measured the levels of DNA polymerase a, p, and y in the S phase of synchronized HeLa cells. They found a rise in the a-polymerase activity while the cells progressed through the S phase, which continued after DNA synthesis had ceased. A concomi tant increase in the y-polymerase level was noted in the early part of the S phase, whereas the Il-polymerase levels remained constant throughout the S phase. Chiu & Baril (130) extended this approach by examining- the complete cell cycle in synchronized HeLa cells. They observed that the a-polymerase activity started to increase in the G1 period before the S phase and that this marked rise in the a-polymerase level Was abolished by addition of cycloheximide, indicating that protein synthesis was necessary for the increase in enzymatic activity. More importantly, the rise in a polymerase activity was observed in the synchronized cell cycle even if DNA synthesis was blocked with hydroxyurea. Since it is known that hydroxyurea does not prevent synchronized G1 cells from entering the S phase (131), the finding that the rise and fall in DNA polytnerase a activity is apparently independent of DNA synthesis signifies that there is no simple

correlation between DNA synthesis and DNA polymerase levels. Further more, the control of DNA synthesis is probably far too subtle to depend solely on DNA polymerase levels, and one should be cautious in interpret ing changes in enzyme activities as being indicative of a necessary role (or lack of it) in the DNA replication process. The rise in a-polymerase activity in growing cells or during the S phase of the cell cycle has been generally assumed to indicate that DNA pOlymerase a is the "replicative" polyme rase in DNA synthesis, but this association may have other causes. Human lymphocytes have provided a useful system for studying changes in DNA polymerases since it is known that stimulation of blood lym phocytes with the plant mitogen phytohemagglutinin (PHA) increases total DNA polymerase activity in these cells (132, 133). Mayer et al (134) have studied the induction of DNA polymerases a, p, and y in this system and



41

found a rise in the activity of all of these enzymes by 72 hr after PHA stimulation of the cells. Interestingly enough, the levels of the {3- and "/ polymerases in the PHA-stimulated cells were quite high-75-90% that of the a-polymerase. The variations in DNA polymerases a and {3 during prolonged stimulation of human lymphocytes have also been examined by Bertazzoni et al (135) and Pedrali Noy et al (136). These workers noted two waves of DNA polymerase induction: the first parallels an increase in DNA replication occurring about 4-5 days after PHA stimulation, and the second wave starts 7-8 days after DNA synthesis has peaked. DNA polymerase a increases sharply during the first wave and drops to low levels during the second wave, while the /3-polymerase rises throughout the first wave of polymerase induction and maintains its level, more or less, during the period from 7 to 12 days after PHA stimulation. On the basis of parallel studies done with ultraviolet light-treated lymphocytes, using hydroxyurea blocking to measure repair DNA synthesis, Bertazzoni et al suggest that the {3-enzyme seems correlated with the ability of cells to perform a DNA repair-type synthesis. These are, obviously, tentative conclusions.

DNA Polymerases during Development Analogous studies have been carried over to differentiating systems in an attempt to find a pattern in the changes in DNA polymerase activity that occur as cells become specialized or mature. Only fragmentary information is available at present, and spermatogenesis has provided one of the systems to study for changes in DNA polymerase during cell development. Chevail lier & Philippe (137) and Hecht et al (138) have examined developing mouse testes and found, respectively, an increase in nuclear /3-polymerase activity during meiotic prophase and a diminution in a-polymerase during the attainment of sexual maturity. Sherman & Kang (139) have measured the DNA polymerase a and {3 levels in midgestation mouse embryo, tropho blast, and decidua. The enzyme levels were found to be highest in the rapidly dividing embryonic cells and lowest in the nonreplicating decidual cells. Changes in DNA polymerase activity have been followed during sea urchin development (140, 141) and in Xenopus laevis (142-144) where there is a noticeable increase in polymerase activity upon maturation of the eggs. In earlier work Chiu & Sung (145) measured changes in two DNA polymerases, which resemble the a- and /3-polymerases, during the devel opment of rat brain and found different patterns in the cerebellum and cortex. Barton & Yang (146) have followed the activities of DNA polyme rases a and {3 in spleens of Balb/c mice during aging. They found no change in the a-polymerase activity in old mice, whereas there was a decrease in the {3-polymerase activity in the spleens of senescent animals compared

42

WEISSBACH

with that of younger animals. Extension of this approach to other aging systems is to be expected. DNA polymerase activity in normal and mal nourished rat placentas has also been measured and correlated with DNA synthesis (147).


Template Studies The template utilization and preferences of the cellular DNA polymerases have been used for identification purposes and examined in hope of obtain ing an insight into the role of these enzymes. Since DNA polymerase activity in vitro, as we know it, is dependent on a 3'-OH priming end as an initiator and a template strand to copy, molecules lacking this juxtaposition cannot be copied, as a general rule. Thus, a fully duplexed DNA strand, linear or circular, is a poor template, as is single-stranded DNA. Studies using these templates must be carefully evaluated to determine if accessory nucleases are present which "activate" the DNA and therefore become the rate-limiting step in the overall reaction. The use of synthetic DNA or RNA templates has been widespread because proper oligomer-homopolymer combinations can be very effective templates. For instance, synthetic ribohomopolymers such as poly(A), in the presence of the proper primer, were found to be exceptionally valuable in the study of the RNA-dependent DNA polymerases (148-50) and provide one of the means of identifying the 'Y-polymerase. Chang & Bollum have done a comprehensive study of various synthetic oligonucleotide-polynucleotide combinations as templates for DNA polymerases a and /3 (7,52). They found that DNA polymerase a seemed most specific for deoxyribose templates such as poly(dA) and could use both oligoribo- and oligodeoxyribonucleotide primers. The /3-polymerase, which could use both polyribo- and polydeoxyribonucleotide templates, such as poly(A) and poly(dA), had a strong preference for oligodeoxyribonucleo tide primers. It should be emphasized that the copying of synthetic ribohomopolymers by the /3- and 'Y-polymerases probably reflects the spe cial configuration of these hompolymers, which can be recognized and read by the enzymes, since neither the a-, /3-, or 'Y-polymerases can copy a natural RNA to any significant extent. Primer and template studies using natural RNA and DNA have been made by Spadari & Weissbach (151). Employing HeLa DNA, which had a short RNA primer molecule hybridized to it, they found that only DNA polymerase a was able to synthesize DNA covalently bound to the RNA (RNA-DNA). Once the RNA-DNA molecule was formed, further exten sion of the DNA chain could be achieved by either the a-, /3- or 'Y polymerases. Their conclusion, in agreement with that of Bollum and Chang, is that the a-polymerase is capable of both RNA- and DNA-primed


43

DNA synthesis, but DNA polymerases /3 and 'Y are capable of only DNA primed synthesis.


Other DNA Polymerases This discussion has centered on work done with the mammalian DNA polymerases. The effort in eukarybtic DNA polymerases has extended well beyond these animals. Although no attempt is made in this article to evalu ate the numerous other studies, the reader may find the following selections useful as a guide to diverse work done in both mammalian and other eukaryotic systems. Two DNA polymerases, which seem to resemble superficially the animal a- and 'Y-polymerases, have been separated from wheat germ and partially characterized (62, 152). McLennan & Keir have identified two major DNA polymerases in Euglena gracilis, purified them (153, 154), and studied their molecular heterogeneity (155) and changes during various growth stages (156). Crown gall tumor cells of periwinkle contain a high-molecular weight DNA polymerase (105,000 daltons), which is sensitive to N-ethyl maleimide but is not inhibited by antibody to the calf thymus a-polymerase (157). Dictyostelium discoideum is reported to have only one DNA polyme rase of 127,000 daltons, which is also inhibited by N-ethylmaleimide (158). Some murine cells have been found to contain a DNA polymerase activ ity that is different from the other three cellular DNA polymerases (159162) and seems to resemble an RNA-dependent DNA polymerase. Bauer & Hofschneider (163) have found a particle-bound RNA-dependent DNA polymerase in the allantoic fluid of uninfected,leukosis virus-free eggs. This enzyme is different from any of the known viral reverse transcriptases and the cellular DNA polymerases a, /3, and 'Y, and may oe similar to the enzymatic activity described by Panet et al (164). In human tissue there have been investigations of DNA polymerase activity in rheumatoid synovial membranes (165, 166) and in human prostatic tissue (167). Finally, there have been two reports of possible new DNA polymerases in human KB cells (168) and human leukemic cells (169).

CONCLUSION Our knowledge of the eukaryotic DNA polymerases is still in the descrip tive stage. Most of the investigations have involved animal cells, tissues, and virus-infected tissue culture cells. Detailed studies in other organisms and biological systems that offer advantages not seen in mammalian systems should be important. For instance, the lack of pertinent mutants and ade quate genetics has made an understanding of the function of vertebrate DNA polymerases a difficult task. Lower eukaryotes such as yeast or the


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basidomycetes could offer powerful genetic approaches to cOIl!plement the DNA polymerase studies already carried out (170-173). Since the DNA polymerase activities in these organisms seem to differ significantly from those of mammalian cells, they may represent a bridge between the proka ryote enzymes and those in higher organisms. It is worth repeating that finding the role of the DNA polymerase in DNA replication will most likely require a knowledge of the role of acces sory factors such as histones. unwinding proteins. or other chromatin pro teins in the overall process. With the identification of the eukaryotic DNA polymerases and their sibling proteins, the enzymatic reconstruction of the steps in DNA replication could begin. Literature Cited 1 . Weissbach. A., Baltimore, D., Bollum, F. J., Gallo, R. C., Kom, D. 1975. Science 1 90:401-2 2. Weissbach, A., Baltimore, D., Bollum, F. J., Gallo, R. C., Kom, D. 1975. Eur. J. Biochem. 59: 1 -2 3. Temin, H. M., Baltimore, D. 1 972. Adv. Virus Res. 1 7 : 1 29-86 4. Wu, A. M., Gallo, R. C. 1 975. In Criti cal Reviews in Biochemistry, ed. G. D. Fasman, pp. 289-347. Cleveland, Ohio:

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Eukaryotic DNA polymerases.

Nomenclature of eukaryotic DNA polymerases.

Translesion Synthesis of 2'-Deoxyguanosine Lesions by Eukaryotic DNA Polymerases.

Isolation and assay of eukaryotic DNA-dependent RNA polymerases.

Isolation and characterization of DNA polymerases from eukaryotic cells.

Eukaryotic nuclear RNA polymerases.

Subunits shared by eukaryotic nuclear RNA polymerases.

Evolution of replicative DNA polymerases in archaea and their contributions to the eukaryotic replication machinery.

Transcription of nucleosomal DNA in SV40 minichromosomes by eukaryotic and prokaryotic RNA polymerases.

Reconstitution of a eukaryotic replisome reveals the mechanism of asymmetric distribution of DNA polymerases.

Yeast DNA polymerases.

DNA polymerases in biotechnology.

DNA polymerases of eukaryotes.

FAM46 proteins are novel eukaryotic non-canonical poly(A) polymerases.

Transcription of chromatin with prokaryotic and eukaryotic polymerases.

Animal cell DNA polymerases in DNA repair.

DNA polymerases of anucleated cells. Isolation and characterization of two DNA polymerases from human platelets.

The DNA polymerases of Chinese hamster cells. Subcellular distribution and properties of two DNA polymerases.

Eukaryotic DNA polymerase ζ.

Eukaryotic DNA replication.

Analysis of nucleotide insertion opposite 2,2,4-triamino-5(2H)-oxazolone by eukaryotic B- and Y-family DNA polymerases.

Eukaryotic DNA helicases.

Eukaryotic DNA replication complex.

Eukaryotic DNA ligases.