of DNA polymerase delta. It has now been discovered that this enzyme also possesses properties clearly distinct from those of DNA polymerase delta. Therefore, this newly discovered enzyme, previously also called DNA polymerase delta-2 o r PCNA-independent DNA polymerase delta, has now been reclassified as DNA polymerase epsilon(728). Its properties and possible biological role are reviewed and discussed in this article.
Summary DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'45'exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, 111 and 11, respectively. Like DNA polymerases I and 111, DNA polymerase I1 is also essential for the viability of cells, which suggests that DNA polymerase I1 (and epsilon) may play a role in DNA replication.
The Catalytic Properties of DNA Polymerases Delta and Epsilon are Different An up-to-date review article about the biochemical properties and role of DNA pol merase delta was recently published in this seriedh4'and will be only briefly cited here. DNA polymerase delta was discovered in 1976, after isolation from rabbit bone marrow, and was defined as a mammalian DNA polymerase with an intrinsic exonuclease activity('). A similar enzyme has since been purified from other sourced4). Excluding readily detectable exonuclease activity, DNA polymerase delta has many catalytic properties in common with DNA polymerase alpha, including sensitivity to inhibition by aphidicolin, N-ethylmaleimide and arabinosyl nucleotides, together with a relative resistance to inhibition by dideoxynucleotides. There are, however, also distinguishing features: DNA polymerase delta is much more resistant to inhibition by the nucleotide analogs butylphenyldGTP and b~tylanilino-dATP(~*~). The two DNA polymerases are also immunologically distinct('"%") and, furthermore, the peptide-mayping patterns of their catalytic subunits are distinct( '). DNA polymerase delta does not efficiently utilize the template/primer poly(dA) .oligo(dT) with long singlestranded areas, except in the presence of proliferating cell nuclear antigen (PCNA , which increases the processivity of the enzyme(12-' ). PCNA is a replication factor that has been inde endently purified first as a cell cycle dependent then as a DNA polymerase delta auxiliary factor(12) and finally, as a factor required for in v i m replication of SV40 DNA('6). A second enzyme that had a readily detectable exonuclease activity, was sensitive to inhibition by aphidicolin and N-ethylmaleimide and by a monoclonal antibody against DNA polymerase alpha, and that was relatively resistant to inhibition by butylphenyl-dGTP and butylanilino-dATP, thus sharing the properties of DNA polymerase delta was first regarded as a form of DNA polymerase delta117-19).The enzyme had, however, also distinguishing catalytic properties, e.g. the efficient utilization of poly(dA) oligo(dT) template/primer with high processivity in the absence of PCNA and relative resistance to inhibition by the nucleotideanalog carbonyldiphosphonate. Also, this enzyme was inhibited by dimethyl sulfoxide (DMSO), a compound that activates DNA polymerase delta(73'9-21).DNA polymerase epsilon has, so far, been purified at least from calf t h y m u ~ ( l ~and 3 ~ ~HeLa ) cells(1s~21). The first 2
1
Introduction Lately, the progress in understanding chromosomal DNA replication in mammalian cells has been rapid. The development of a cell-free in v i m replication system for SV40 DNA, which has allowed the purification of numerous cellular replication factors (reviewed in ref. l), and the characterization of novel replication factors including DNA polymerases, have greatly contributed to this progress. So far, five types of DNA polymerases have been defined in mammalian cells: DNA polymerases alpha, beta, gamma, delta and epsilon (reviewed in refs 2-6). DNA polymerase beta is possibly required for DNA repair, DNA polymerase gamma for the replication of mitochondria1 DNA, and DNA polymerases alpha and delta for the replication of chromosomal DNA. Readily detectable, tightly associated 3'+5' exonuclease activity. is typical for DNA polymerase delta. It is no wonder, that a second DNA polymerase with 3'+5' exonuclease activity and with an inhibitor spectrum very similar to that of DNA polymerase delta was, until recently, regarded as a form
-
delta-like DNA polymerase from rabbit bone marrow(') was also able to use poly(dA). oligo(dT) template/primer(22)and was possibly DNA polymerase epsilon. The response of that enzyme to PCNA has not, however, been studied. This is also the case with an enzyme from human placenta(23).
common. Peptide mapping of the large subunits of the two enzymes and of DNA polymerase alpha, purified from the same HeLa extract, confirmed that all three DNA polymerases are structurally distinct enzymes(7). The distinguishing features between DNA polymerases delta and epsilon are summarized in Table 1.
The Polypeptide Structures of DNA Polymerases Delta and Epsilon are Distinct If DNA polymerases delta and epsilon were distinct enzymes, it should be possible to purify both the enzymes from the same source. It turned out that DNA polymerases delta and epsilon could be easily separated from each other by conventional column chromatography and the two enzymes as well as DNA polymerase alpha were recently purified to apparent homo eneity simultaneously from the same HeLa extract($. Calf thymus(24)and HeLa DNA polymerase delta(7) is a heterodimer of 125-130 and 47-48 kDa subunits. Immunoblotting with a neutralizing antibody 'suggests that the olymerase activity is located in the larger subunit('' . DNA polymerase epsilon has been purified from calf thymus as a preparation consisting of 140,125, 48 and 40 kDa polypeptides(2s).Polymerase activity was assigned to the 140 and 125kDa polypeptides by localizing the activity after renaturing the polypeptides on an SDS-polyacrylamide gel. However, these active polypeptides are probably proteolytic fragments from a larger form since the enzyme from HeLa cells has two subunits of 215 and 55kDac21),the larger of which possesses the polymerase activity(26). An alternative explanation would be different splicing of RNA. A second preparation from calf thymus had several polypeptides, the sizes of which ranged from 45 to 245 kDa(17), but the activity was not located. A polyclonal antibody raised against calf thymus DNA polymerase delta neutralizes HeLa DNA polymerase epsilon activity, but does not recognize any polypeptides in immunoblotting("). This behavior suggests that the structures of the two enzymes are distinct, but that they may share some structural features in areas that are essential for polymerase activities. Comparison of the active subunits of DNA polymerase delta from calf thymus and of DNA polymerase epsilon from HeLa cells supports this hypothesis. The peptide mapping patterns are essentially different, but may have some peptides in
Mammalian DNA Polymerase Epsilon is a Counterpart of Yeast DNA Polymerase II As described, there is strong evidence that DNA polymerases delta and epsilon are distinct enzymes. This argument is based on comparative peptide maps, distinguishing immunologic and catalytic properties as well as chromatographic behavior. Comparison with yeast DNA polymerases gives further support to this argument. DNA polymerases I, I1 and 111 from yeast cells are counterparts of mammalian DNA polymerases alpha, epsilon and delta, respectively. The similarities in physical and catalytic properties between yeast and mammalian DNA polymerases are striking, suggesting strong conservation of these enzymes. The amino acid se uence homology between yeast DNA 01 merase I(2 ) and human DNA polymerase alpha(') :atalytic peptides is 31 %(29).In addition, the subunit structure, includin the molecular weights of associated polypeptided3', ), is conserved with a remarkable accuracy. Yeast DNA polymerase 111, like mammalian DNA polymerase delta, is activated and made processive by both yeast and mammalian PCNA(32*33)and the two enzymes have very similar inhibitor The molecular weight of the yeast enzyme, as calculated from the cDNA sequence, is 125 kDa(34).This size is comparable to the molecular weight of 125-130 kDa estimated for the corresponding mammalian enzyme on SDS-polyacrylamide gel^(^*^^). Yeast DNA polymerase I1 is a hi hly rocessive enzyme and not activated by PCNA(323 ), properties in common with DNA polymerase epsilon. Both enzymes are very active when poly(dA).oligo(dT) is used as a template/primer and both can be activated to use DNase I-activated DNA by KCI concentrations corresponding to physiological ionic strengths. The inhibitor specificities of the two enzymes are also identica1(17-19.21 ,3S-38). Yeast DNA polymerases I, I1 and 111 are known to be encoded by different genes, which have been cloned and s e q ~ e n c e d ( ~ ~ ,The ~ ~ ~calculated ~'). molecular weight of DNA polymerase I1 is 256 kDa,
P
9
4
g
Table 1. Distinguishing properties of DNA polymerases delta and epsilon
Property
Pol delta
Pol epsilon
Subunits (kDa) Catalytic polypeptide Preferred template/primer Processivity Processivity increased by PCNA Effect of DMSO Inhibition by carbonyldiphosphonate Recognition by anti-pol delta in immunoblotting Peptide maps
125, 47 12s Poly(dA-dT)
>200,55
>2w Poly(dA). oligo(dT) High
Low Yes Activates Strong Yes
?
Inhibits Weak No Distinct
p
which is close to the molecular weight of 215 kD of the catalytic peptide of the HeLa DNA polymerase epsilon(21). The fact that both DNA polymerase and 3'+5' exonuclease motifs can be found in the amino acid sequence of the yeast DNA polymerase 11(39) further suggests that both of these activities are probably located in the large subunit of DNA polymerase epsilon. The amino-terminal portion of the 256 kDa yeast enzyme has both DNA polymerase and exonuclease motifs, while the C-terminal part may be required for the association of smaller polypeptides found in yeast enzyme preparations(39). The enzyme has been purified from yeast extract as a 200kDa polypeptide together with four smaller polypeptides, as a 145 kDa polypeptide(35) and as a 132 kDa polypeptide(38). This variation may result from proteolytic cleavage either in vivo or during purification, as suggested above as probably being the case with DNA polymerase epsilon. DNA Polymerase Epsilon is a Repair Enzyme DNA polymerase epsilon has been isolated from HeLa cells as a factor required for restoring DNA synthesis activit in UV-irradiated permeabilized fibrob l a s t ~ ~ ~I~n ~this ' ) .process, DNA polymerase epsilon was obviously required for DNA synthesis, since DNA polymerase alpha or beta alone could not promote synthesis; nor could synthesis be prevented by specific inhibitors for DNA polymerase alpha. The fact that DNA polymerase alpha is inactive in promoting repair synthesis alone, but increases synthesis when added together with DNA polymerase epsilon, suggests that epsilon is required at least for the initiation of repair synthesis, after which DNA polymerase alpha could participate in the process. Numerous reports have been published about attempts to estimate the roles of different DNA polymerases in DNA repair synthesis by using permeabilized cells or cell extracts. Earlier, when these studies were usually planned to distinguish between DNA polymerases alpha and beta, DNA polymerases delta and epsilon were unknown. Recently, the participation of DNA polymerase delta has also been considered. The existence of DNA polymerase epsilon, however, has not been taken into account and these studies should certainly be re-evaluated. The yeast counterpart of DNA polymerase epsilon, DNA polymerase 11, has also been su gested to be involved in DNA repair and mutagenesid3 ,but no direct evidence for the participation of these functions has been presented.
6
Is DNA Polymerase Epsilon Involved in DNA Replication? Many lines of evidence suggest that DNA polymerase alpha and delta, and their counterparts in yeast cells, DNA polymerases I and 111, are re uired for the . A replication of chromosomal DNA". 1 according to which DNA polymerase 36,28341-43)
alpha carries out lagging-strand synthesis while DNA polymerase delta is responsible for leading-strand synthesis is consistent with the properties of the purified enzymes. DNA polymerase alpha has low processivity and is usually purified together with a primase, the properties that fit well the requirements for frequent priming events and subsequent filling of relatively short single-stranded stretches on lagging strands. Highly processive DNA polymerase delta in the presence o f PCNA could be needed for the synthesis of long, noninterruped stretches of DNA on leading strands. This model is also supported by evidence from the studies of SV40 DNA replication in v i m . In this system, PCNA seems to be required for leading-strand synthesis since in its absence only short nascent lagging-strand fragments were synthesized(4s).The direct requirement of DNA polymerase delta for the initiation of leadingstrand synthesis and DNA polymerase alpha for the initiation of lagging-strand s nthesis has also been demonstrated in this system (467. DNA polymerase epsilon has been implicated in DNA repair("). DNA polymerase epsilon is an inherently processive enzyme, the property that could be expected for an enzyme responsible for the bulk of DNA synthesis in chromosomal DNA replication. This raises a question of whether it is also involved in DNA replication. Indeed, it was recently found that the inactivation of the gene coding for yeast DNA polymerase 11, a counterpart of mammalian DNA polymerase epsilon, resulted in inviability of cells(39). Repair genes that have been identified in yeast cells are not essential for the viability of yeast cells, with the exception of RAD3(47).However, the essential function of this gene may not be repair since several missense mutations in this gene result in the complete loss of excision repair but not in the loss of viability. A defect in repair function is thus not a likely reason for the loss of viability of cells harboing mutations in the gene coding for DNA polymerase 11, but rather a defect in replication. Furthermore, these mutant cells show the dumb-bell terminal morphology which is characteristic for the arrest of DNA replication. A putative regulatory sequence for replication genes(27)also occurs in the 5' non-coding region of the gene. The above data suggest that, in addition to DNA polymerases I and 111, DNA polymerase I1 may also be required for the replication of chromosomal DNA in yeast cells. Considering striking conservation of the properties of DNA polymerases in eukaryotes, DNA polymerase epsilon may be, by analogy with yeast DNA polymerase 11, required for replication in mammalian cells. If DNA polymerase I1 (epsilon) is involved in DNA replication, the next question to be asked concerns its role in the process. Does it play a specific role at replication forks together with DNA polymerases I (alpha) and I11 (delta), or is it required for the replication of certain special areas in chromosomes? A model with roles for all three DNA olymerases at a replication fork has been presentedk'). As yet, this question has not, however, been addressed at the
experimental level and discussion of this model is beyond the scope of this review.
Acknowledgements The author’s research is supported by a grant from The Academy of Finland. References 1 KELLY,T. J . (1988). SV40 DNA Replication. J. Bid. Chem. 263,
17 889-17 892. 2 FRY,M. A N D LOEB,L. A. (1986). AnimalcellDNA polymerases. CRC Press,
Boca Raton, Florida. 3 KAGUNI, L. S. AND LEHMAN, I . R. (1988). Eukaryotic DNA polymeraseprimase: structure. mechanism and function. Biochem. Biophyi. Actu 950. 87-101. 4 BURGERS, P. M. J. (1989). Eukaryotic DNA polymerases alpha and delta: conserved properties and interactions from yeast to mammalian cells. Prog. Nucl. Acid Res. Mol. Biol. 37, 235-258. 5 LEHMAN. I. R. AND KAGUNI, L. S. (1989). DNA polymerase alpha. J. B i d . Chem. 264. 4265-4268. 6 DOWNEY, K. M.. TAN,C.-K. A N D So, A. G. (1990). DNA polymerase delta: a second eukaryotic DNA replicase. BioEssays 12, 231-236. 7 SYVAOJA. J.. SUOMENSAARI. S . . NISHIDA. C., GOLDSMITH, J. S . , CHUI.G., JAIN. S. A N D LINN,S. (1990). DNA polymerases alpha, delta and epsilon: three distinct enzymes from HeLa cells. Proc. Natl Acad. Sci. USA 87. 6664-6668. 8 BURGERS, P. M. J . , BAMBARA, R. A,. CAMPBELL, J. L., CHANG.L. M. S . , DOWNEY, K. M., HUBSCHER, U.. LEE,M. Y. W. T., LINN,S. M., So, A. G . A N D SPADARI, S. (1990). Revised nomenclature for eukaryotic DNA polymerases. Eur. J. Biochem., 191, 617-618. 9 BYRNES, J . J.. DOWNEY. K. M.. BLACK. V. L. A N D So. A. G. (1976). A new mammalian DNA polymerase with 3’ to 5’ exonuclease activity: DNA polymerase delta. Biochemistry 15. 2817-2823. 10 DOWNEY, K. M.. TAN,C.-K.. ANDREWS, D. M., LI, X. AND So, A. G. (1988). Proposed roles for DNA polymerases alpha and delta at the replication fork. Cancer Cells 6 , 403-410. 11 WONG,S. W., SYVAOJA, J . . TAN,C.-K., DOWNEY, K. M., So, A. G., LINN, S. AND WANG, T. S.-F. (1989). DNA polymerase alpha and delta are immunologically and structurally distinct. J. Biol. Chem. 264, 5924-5928. 12 TAN,C.-K., CASTILLO, C.. So. A. G. A N D DOWNEY, K. M. (1986). An auxiliary protein for DNA polymerase delta from fetal calf thymus. J. B i d . Chem. 261. 12310-12316. 13 PRELICH. G . , TAN,C.-K., KOSTURA, M., MATTHEWS, M. B., So, A. G.. DOWNEY, K. M. AND STILLMAN, B. (1987). Functional identity of proliferating cell nuclear antigen and a DNA polymerase delta auxiliary protein. Nature 326, 517-520. 14 BRAVO. R. AND CELIS,J. E. (1980). A search for differential polypeptide synthesis throughout the cell cycle of HeLa cells. J. Cell Biol. 84, 795-802. 15 MATTHEWS. M. B., BERNSTEIN, R.M., FRANZA, B. R . , JR AND GARRELS. J. 1. (1984). Identity of proliferating cell nuclear antigen and cyclin. Nature 309. 374-376. 16 PRELICH. G.. KOSTURA. M.. MARSHAK. D. R . , MATTHEWS. M. B. AND STILLMAN, B. (1987). The cell-cycle regulated proliferating cell nuclear antigen is required for SV40 DNA replication in vifro. Nature 326, 471-475. 17 CRUTE, J . J.. WAHL,A. F. A N D BAMBARA, R. A . (1986). Purification and characterization of two new high molecular weight forms of DNA polymerase delta. Biochemistry 25, 26-36. 18 NISHIDA, C.. REINHARD, P. AND LINN,S . (1988). DNA repair synthesis in human fibroblasts requires DNA polymerase delta. J. Biol. Chem. 263, 501-510. 19 FOCHER, F.. SPADARI. S . , GINELLI. B.. HOTTIGER, M., GRASSMANN, M. A N D HUBSCHER. U. (1988). Calf thymus DNA polymerase delta: purification, biochemical and functional properties of the enzyme after its separation from DNA polymerase alpha, a DNA dependent ATPase and proliferating cell nuclear antigen. Nucl. Acids Res. 14, 6279-6295. 20 SABATINO. R. D.. MYERS.T. W.. BAMBATA. R. A , , OHOAK.K.-S.. MARACCINO. R . L. A N D FRICKEY. P. H. (1988). Calf thymus DNA polymerase alpha and delta are capable of highly processive DNA synthesis. Biochemistry 27, 2998-3004. 21 SYVAOJA, J . E . A N D LwN. S. (1989). Characterization of a large form of DNA polymerase delta from HeLa cells that is insensitive to proliferating cell nuclear antigen. J. Biol. Chent. 264, 2489-2497. 22 BYRNES, J. J. A N D BLACK, V. L. (1978). Comparison of DNA polymerase alpha and delta from bone marrow. Biochemistry 17. 4226-4231. 23 LEE, M. Y . W . T. AND TOOMEY, N. L. (1987). Human placental DNA polymerase delta: identification of a 170-kilodalton polypeptide by activity staining and immunoblotting. Biochemistry 26. 1076-1085. 24 LEE, M. Y. W. T., TAN,C.-K., DOWNEY, K. M. AND So, A. G. (1984).
Further studies on calf thymus DNA polymerase delta purified to homogeneity by a new procedure. Biochemkrry 23, 1906-1913. 25 FOCHER, F., GASSMANN, M., HAFKEMEYER. P . . FERRARI. E.. SPADARI. S. A N D HUBSCHER, U . (1989). Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA). Nucl. Acids Res. 17, 1805-1821. 26 KESTI.T. AND SYVAOJA, J. E. (1990). Identification and tryptic cleavage of the catalytic core of HeLa and calf thymus DNA polymerase epsilon. Submitted for publication. 27 PIZZAGALLI, A., VALSASNINI, P., PLEVANI. P. A N D LUCCHINI, G. (1988). DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation. and protein homology with other DNA polymerases. Proc. Natl Acad. Sci. USA 85. 3712-3776. 28 WONG.S. W., WAHL.A. F., YUAN,P.-M.. ARAI.N., PEARSON, B. E., ARAI, K.-I.. KORN.D.. HUNKAPILLER, M. W. A N D WANG,T . S.-F. (1988). Human DNA Polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases. EMBO J. 7, 37-47. 29 WANG,T. S.-F.. WONG,S. W. A N D KORN. D. (1989). Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases. FASEB 3, 14-21. 30 PLEVANI. P . . FOIANI, M., VALSASNINI. P.. BADARACCO.G . , E. AND CHANG,L. M. S. (1985). Polypeptide structure of CHERIATHUNDAM, DNA primase from a yeast DNA polymerase-primase complex. J. Biol. Chem. 260, 7102-7107. 31 WONG,S. W., PADORSKY, L. R., FISCHER, P. A , , WANG,T. S.-F. A N D KORN. D. (1986). Structural and enzymological characterization of immunoaffinitypurified DNA polymerase alpha-primase complex from KB cells. J. B i d . Chern. 261. 7958-7968. 32 BAUER.G. A. AND BURGERS, P. M. J . (19x8). The yeast analog of mammalian cyclin/proliferating-cell nuclear antigen interacts with mammalian DNA polymerase delta. Proc. Natl Acad. Sci. USA 85. 7506-7510. 33 BURGERS, P. M. J . (1988). Mammalian cyclin/PCNA (DNA polymerase delta auxiliary protein) stimulates processive DNA synthesis by yeast DNA polymerase 111. Nucl. Acids Res. 16, 6297-6307. 34 BOULET, A., SIMON, M., FAYE,G.. BAUER,G. A. A N D BURGERS, P. M. J. (1989). Structure and function of the Saccharomyces cerevisiae CDC2 gene encoding the large subunit of DNA polymerase 111. EMBO J. 8, 1M9-1854. 35 HAMATAKE, R . K . . HASEGAWA, H., CLARK. A. B., BEBENEK, K.. KUNKEL, T. AND SUGINO,A. (1990). Purification and characterization of DNA polymerase 11 from the yeast Saccharomyce$ cerevisiue. J. Biol. Chem. 265, 4072-4083. 36 WINTERSBERGER, E. (1974). Deoxyribonucleic acid polymerase from yeast: further purification and characterization of DNA-dependent DNA polymerases A and B. Eur. J. Biochem. 50, 41-47. 37 CHANC. L. M. (1976). DNA polymerase from Bakers’ yeast. J. Biol. Chem. 252. 1873-1880. 38 BUDD.M. E. . SITNEY, K. C. A N D CAMPBELL, J. L. (1989). Purification of DNA polymerase 11. a distinct DNA polymerase, from Sucrharomyces cerevisiae. J. Biol. Chem. 264, 6557-6565. 39 MORRISON, A , , ARAKI, H., CLARK, A . B., HAMATAKE, R. K. AND SUGINO. A. (1990). A third essential DNA polymerase in Saccharomyces cerevisiae. Cell 62, 1143-1151. 40 NISHIDA, C. AND LINN,S. (1988). DNA repair synthesis in permeabilized human fibroblasts mediated by DNA polymerase delta and application for purification of xeroderma pigmentosum factors. Cancer cells 6 , 411-415. 41 WEINBERG. D. H. A N D KELLY,T. J. (1989). Requirement for t w o DNA polymerases in the replication of simian virus 40 DNA in v i m . Proc. Natl Acad. Sci. USA 86, 9742-9746. 42 TSURIMOTO. T. AND STILLMAN, B. (1989). Multiple replication factors augment DNA synthesis by two eukaryotic DNA polymerases. alpha and delta. E M B O J. 8, 3883-3889. 43 LEE. S.-H., EKI,T. A N D HURWITZ, J. (1989). Synthesis of DNA containing the simian virus origin of replication by the combined action of DNA polymerases alpha and delta. Proc. Natl Acad. Sci. USA 86, 7361-7365. 44 So, A. G. AND DOWNEY, K. M. (1988). Mammalian DNA polymerases alpha and delta: current status in DNA replication. Biochemistry 27. 4591-4595. 45 PRELICH, G . AND STILLMAN, B . (1988). Coordinated leading and lagging strand synthesis during SV40 DNA replication in virro requires PCNA. Cell53. 117-126. 46 TSURIMOTO, T.. MELENDY, T. A N D STILLMAN, B. (1990). Sequential initiation of lagging and leading strand by two different polymerase complexes at the SV40 DNA replication origin. Nature 346, 534-539. 47 FRIEDBERG, E . C. (1988). Deoxyribonucleic acid repair in the yeast Succhoromyces cerevisiae. Microbiol. Rev. 52, 70-102.
Juhani E. Syvaoja is at the Department of Biochemistry, University of Oulu, Linnanmaa SF-90570 Oulu, Finland.
Summary DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'45'exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, 111 and 11, respectively. Like DNA polymerases I and 111, DNA polymerase I1 is also essential for the viability of cells, which suggests that DNA polymerase I1 (and epsilon) may play a role in DNA replication.
The Catalytic Properties of DNA Polymerases Delta and Epsilon are Different An up-to-date review article about the biochemical properties and role of DNA pol merase delta was recently published in this seriedh4'and will be only briefly cited here. DNA polymerase delta was discovered in 1976, after isolation from rabbit bone marrow, and was defined as a mammalian DNA polymerase with an intrinsic exonuclease activity('). A similar enzyme has since been purified from other sourced4). Excluding readily detectable exonuclease activity, DNA polymerase delta has many catalytic properties in common with DNA polymerase alpha, including sensitivity to inhibition by aphidicolin, N-ethylmaleimide and arabinosyl nucleotides, together with a relative resistance to inhibition by dideoxynucleotides. There are, however, also distinguishing features: DNA polymerase delta is much more resistant to inhibition by the nucleotide analogs butylphenyldGTP and b~tylanilino-dATP(~*~). The two DNA polymerases are also immunologically distinct('"%") and, furthermore, the peptide-mayping patterns of their catalytic subunits are distinct( '). DNA polymerase delta does not efficiently utilize the template/primer poly(dA) .oligo(dT) with long singlestranded areas, except in the presence of proliferating cell nuclear antigen (PCNA , which increases the processivity of the enzyme(12-' ). PCNA is a replication factor that has been inde endently purified first as a cell cycle dependent then as a DNA polymerase delta auxiliary factor(12) and finally, as a factor required for in v i m replication of SV40 DNA('6). A second enzyme that had a readily detectable exonuclease activity, was sensitive to inhibition by aphidicolin and N-ethylmaleimide and by a monoclonal antibody against DNA polymerase alpha, and that was relatively resistant to inhibition by butylphenyl-dGTP and butylanilino-dATP, thus sharing the properties of DNA polymerase delta was first regarded as a form of DNA polymerase delta117-19).The enzyme had, however, also distinguishing catalytic properties, e.g. the efficient utilization of poly(dA) oligo(dT) template/primer with high processivity in the absence of PCNA and relative resistance to inhibition by the nucleotideanalog carbonyldiphosphonate. Also, this enzyme was inhibited by dimethyl sulfoxide (DMSO), a compound that activates DNA polymerase delta(73'9-21).DNA polymerase epsilon has, so far, been purified at least from calf t h y m u ~ ( l ~and 3 ~ ~HeLa ) cells(1s~21). The first 2
1
Introduction Lately, the progress in understanding chromosomal DNA replication in mammalian cells has been rapid. The development of a cell-free in v i m replication system for SV40 DNA, which has allowed the purification of numerous cellular replication factors (reviewed in ref. l), and the characterization of novel replication factors including DNA polymerases, have greatly contributed to this progress. So far, five types of DNA polymerases have been defined in mammalian cells: DNA polymerases alpha, beta, gamma, delta and epsilon (reviewed in refs 2-6). DNA polymerase beta is possibly required for DNA repair, DNA polymerase gamma for the replication of mitochondria1 DNA, and DNA polymerases alpha and delta for the replication of chromosomal DNA. Readily detectable, tightly associated 3'+5' exonuclease activity. is typical for DNA polymerase delta. It is no wonder, that a second DNA polymerase with 3'+5' exonuclease activity and with an inhibitor spectrum very similar to that of DNA polymerase delta was, until recently, regarded as a form
-
delta-like DNA polymerase from rabbit bone marrow(') was also able to use poly(dA). oligo(dT) template/primer(22)and was possibly DNA polymerase epsilon. The response of that enzyme to PCNA has not, however, been studied. This is also the case with an enzyme from human placenta(23).
common. Peptide mapping of the large subunits of the two enzymes and of DNA polymerase alpha, purified from the same HeLa extract, confirmed that all three DNA polymerases are structurally distinct enzymes(7). The distinguishing features between DNA polymerases delta and epsilon are summarized in Table 1.
The Polypeptide Structures of DNA Polymerases Delta and Epsilon are Distinct If DNA polymerases delta and epsilon were distinct enzymes, it should be possible to purify both the enzymes from the same source. It turned out that DNA polymerases delta and epsilon could be easily separated from each other by conventional column chromatography and the two enzymes as well as DNA polymerase alpha were recently purified to apparent homo eneity simultaneously from the same HeLa extract($. Calf thymus(24)and HeLa DNA polymerase delta(7) is a heterodimer of 125-130 and 47-48 kDa subunits. Immunoblotting with a neutralizing antibody 'suggests that the olymerase activity is located in the larger subunit('' . DNA polymerase epsilon has been purified from calf thymus as a preparation consisting of 140,125, 48 and 40 kDa polypeptides(2s).Polymerase activity was assigned to the 140 and 125kDa polypeptides by localizing the activity after renaturing the polypeptides on an SDS-polyacrylamide gel. However, these active polypeptides are probably proteolytic fragments from a larger form since the enzyme from HeLa cells has two subunits of 215 and 55kDac21),the larger of which possesses the polymerase activity(26). An alternative explanation would be different splicing of RNA. A second preparation from calf thymus had several polypeptides, the sizes of which ranged from 45 to 245 kDa(17), but the activity was not located. A polyclonal antibody raised against calf thymus DNA polymerase delta neutralizes HeLa DNA polymerase epsilon activity, but does not recognize any polypeptides in immunoblotting("). This behavior suggests that the structures of the two enzymes are distinct, but that they may share some structural features in areas that are essential for polymerase activities. Comparison of the active subunits of DNA polymerase delta from calf thymus and of DNA polymerase epsilon from HeLa cells supports this hypothesis. The peptide mapping patterns are essentially different, but may have some peptides in
Mammalian DNA Polymerase Epsilon is a Counterpart of Yeast DNA Polymerase II As described, there is strong evidence that DNA polymerases delta and epsilon are distinct enzymes. This argument is based on comparative peptide maps, distinguishing immunologic and catalytic properties as well as chromatographic behavior. Comparison with yeast DNA polymerases gives further support to this argument. DNA polymerases I, I1 and 111 from yeast cells are counterparts of mammalian DNA polymerases alpha, epsilon and delta, respectively. The similarities in physical and catalytic properties between yeast and mammalian DNA polymerases are striking, suggesting strong conservation of these enzymes. The amino acid se uence homology between yeast DNA 01 merase I(2 ) and human DNA polymerase alpha(') :atalytic peptides is 31 %(29).In addition, the subunit structure, includin the molecular weights of associated polypeptided3', ), is conserved with a remarkable accuracy. Yeast DNA polymerase 111, like mammalian DNA polymerase delta, is activated and made processive by both yeast and mammalian PCNA(32*33)and the two enzymes have very similar inhibitor The molecular weight of the yeast enzyme, as calculated from the cDNA sequence, is 125 kDa(34).This size is comparable to the molecular weight of 125-130 kDa estimated for the corresponding mammalian enzyme on SDS-polyacrylamide gel^(^*^^). Yeast DNA polymerase I1 is a hi hly rocessive enzyme and not activated by PCNA(323 ), properties in common with DNA polymerase epsilon. Both enzymes are very active when poly(dA).oligo(dT) is used as a template/primer and both can be activated to use DNase I-activated DNA by KCI concentrations corresponding to physiological ionic strengths. The inhibitor specificities of the two enzymes are also identica1(17-19.21 ,3S-38). Yeast DNA polymerases I, I1 and 111 are known to be encoded by different genes, which have been cloned and s e q ~ e n c e d ( ~ ~ ,The ~ ~ ~calculated ~'). molecular weight of DNA polymerase I1 is 256 kDa,
P
9
4
g
Table 1. Distinguishing properties of DNA polymerases delta and epsilon
Property
Pol delta
Pol epsilon
Subunits (kDa) Catalytic polypeptide Preferred template/primer Processivity Processivity increased by PCNA Effect of DMSO Inhibition by carbonyldiphosphonate Recognition by anti-pol delta in immunoblotting Peptide maps
125, 47 12s Poly(dA-dT)
>200,55
>2w Poly(dA). oligo(dT) High
Low Yes Activates Strong Yes
?
Inhibits Weak No Distinct
p
which is close to the molecular weight of 215 kD of the catalytic peptide of the HeLa DNA polymerase epsilon(21). The fact that both DNA polymerase and 3'+5' exonuclease motifs can be found in the amino acid sequence of the yeast DNA polymerase 11(39) further suggests that both of these activities are probably located in the large subunit of DNA polymerase epsilon. The amino-terminal portion of the 256 kDa yeast enzyme has both DNA polymerase and exonuclease motifs, while the C-terminal part may be required for the association of smaller polypeptides found in yeast enzyme preparations(39). The enzyme has been purified from yeast extract as a 200kDa polypeptide together with four smaller polypeptides, as a 145 kDa polypeptide(35) and as a 132 kDa polypeptide(38). This variation may result from proteolytic cleavage either in vivo or during purification, as suggested above as probably being the case with DNA polymerase epsilon. DNA Polymerase Epsilon is a Repair Enzyme DNA polymerase epsilon has been isolated from HeLa cells as a factor required for restoring DNA synthesis activit in UV-irradiated permeabilized fibrob l a s t ~ ~ ~I~n ~this ' ) .process, DNA polymerase epsilon was obviously required for DNA synthesis, since DNA polymerase alpha or beta alone could not promote synthesis; nor could synthesis be prevented by specific inhibitors for DNA polymerase alpha. The fact that DNA polymerase alpha is inactive in promoting repair synthesis alone, but increases synthesis when added together with DNA polymerase epsilon, suggests that epsilon is required at least for the initiation of repair synthesis, after which DNA polymerase alpha could participate in the process. Numerous reports have been published about attempts to estimate the roles of different DNA polymerases in DNA repair synthesis by using permeabilized cells or cell extracts. Earlier, when these studies were usually planned to distinguish between DNA polymerases alpha and beta, DNA polymerases delta and epsilon were unknown. Recently, the participation of DNA polymerase delta has also been considered. The existence of DNA polymerase epsilon, however, has not been taken into account and these studies should certainly be re-evaluated. The yeast counterpart of DNA polymerase epsilon, DNA polymerase 11, has also been su gested to be involved in DNA repair and mutagenesid3 ,but no direct evidence for the participation of these functions has been presented.
6
Is DNA Polymerase Epsilon Involved in DNA Replication? Many lines of evidence suggest that DNA polymerase alpha and delta, and their counterparts in yeast cells, DNA polymerases I and 111, are re uired for the . A replication of chromosomal DNA". 1 according to which DNA polymerase 36,28341-43)
alpha carries out lagging-strand synthesis while DNA polymerase delta is responsible for leading-strand synthesis is consistent with the properties of the purified enzymes. DNA polymerase alpha has low processivity and is usually purified together with a primase, the properties that fit well the requirements for frequent priming events and subsequent filling of relatively short single-stranded stretches on lagging strands. Highly processive DNA polymerase delta in the presence o f PCNA could be needed for the synthesis of long, noninterruped stretches of DNA on leading strands. This model is also supported by evidence from the studies of SV40 DNA replication in v i m . In this system, PCNA seems to be required for leading-strand synthesis since in its absence only short nascent lagging-strand fragments were synthesized(4s).The direct requirement of DNA polymerase delta for the initiation of leadingstrand synthesis and DNA polymerase alpha for the initiation of lagging-strand s nthesis has also been demonstrated in this system (467. DNA polymerase epsilon has been implicated in DNA repair("). DNA polymerase epsilon is an inherently processive enzyme, the property that could be expected for an enzyme responsible for the bulk of DNA synthesis in chromosomal DNA replication. This raises a question of whether it is also involved in DNA replication. Indeed, it was recently found that the inactivation of the gene coding for yeast DNA polymerase 11, a counterpart of mammalian DNA polymerase epsilon, resulted in inviability of cells(39). Repair genes that have been identified in yeast cells are not essential for the viability of yeast cells, with the exception of RAD3(47).However, the essential function of this gene may not be repair since several missense mutations in this gene result in the complete loss of excision repair but not in the loss of viability. A defect in repair function is thus not a likely reason for the loss of viability of cells harboing mutations in the gene coding for DNA polymerase 11, but rather a defect in replication. Furthermore, these mutant cells show the dumb-bell terminal morphology which is characteristic for the arrest of DNA replication. A putative regulatory sequence for replication genes(27)also occurs in the 5' non-coding region of the gene. The above data suggest that, in addition to DNA polymerases I and 111, DNA polymerase I1 may also be required for the replication of chromosomal DNA in yeast cells. Considering striking conservation of the properties of DNA polymerases in eukaryotes, DNA polymerase epsilon may be, by analogy with yeast DNA polymerase 11, required for replication in mammalian cells. If DNA polymerase I1 (epsilon) is involved in DNA replication, the next question to be asked concerns its role in the process. Does it play a specific role at replication forks together with DNA polymerases I (alpha) and I11 (delta), or is it required for the replication of certain special areas in chromosomes? A model with roles for all three DNA olymerases at a replication fork has been presentedk'). As yet, this question has not, however, been addressed at the
experimental level and discussion of this model is beyond the scope of this review.
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Juhani E. Syvaoja is at the Department of Biochemistry, University of Oulu, Linnanmaa SF-90570 Oulu, Finland.
