DNA Polymerase II, the Epsilon Polymerase of Saccharomyces cerevisiae

DNA Polymerase II, the Epsilon Polymerase of Saccharomyces cerevisiae' ALAN MORRISONAND AKIO sUCIN02 Laboratory of Molecular Genetics National Institute of Enoironmental Health Sciences Research Triangle Park, North Carolina 27709 Categorization and Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . DNA Polymerase I1 Structure and Activities . . . . . . . . . . . . . . . . . . . . . . Cell Cycle Regulation ....... ........... Domain Structure of Catalytic Subunit . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetics of 3'-+5' Exonuclease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The DNA Repair Polymerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Role of DNA Polymerase I1 in DNA Replication . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. 11. 111. IV. V. VI.

94 96 103 104 110

112 114 117

To date, five nuclear D N A polymerases have been observed or posited to exist in Saccharomyces cerevisiae, prompting questions of why there are so many, and what their roles are. Progress toward answering some of these questions has come from the SV40 (simian virus 40) in oitro D N A replication system. Seven cellular factors required for this system (I), including D N A polymerases a and 6 (2-4), have also been detected in S. cerecisiae, enabling genetic tests ofwhether the in vitro system truly reflects in vico chromosomal replication. The purely genetic approach identified a series of celldivision-cycle mutants, among which were alleles of the genes now known to encode the cx and 6 D N A polymerases (5-8). DNA polymerase I1 was detected enzymatically in yeast-cell extracts, but its gene, POL2,3 was not 1 Abbreviations used: SV40, simian virus 40; PCNA, proliferating cell nuclear antigen; UV, ultraviolet; SSB, single-stranded DNA-binding protein; RF-C, replication factor C; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate. To whom correspondence may he addressed. Present address: Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan. A list of yeast gene symhols and an explanation of the conventions used in this essay are given in the Glossary at the end of this review article.

94

ALAN MORRISON AND AKIO SUCINO

found in any genetic screen, nor was its mammalian counterpart identified in the SV40 in vitro system. POL2 was eventually cloned by reverse genetics. In this article, we review data on DNA polymerase I1 in the context of current knowledge of eukaryotic DNA polymerases, DNA replication and its fidelity, and DNA repair. Information on related subjects is covered in reviews of eukaryotic DNA polymerases (9-13), eukaryotic DNA replication (14), yeast DNA replication (15), the SV40 in citro system (16), and exonucleolytic proofreading (17, 18).

1. Categorization and Nomenclature

A. Eukaryotic DNA Polymerases Six genes encoding observed or predicted DNA polymerase activities have been mapped and sequenced in S. cerevisim (Table I). Activities encoded by POLl, POL2, POL.3, and M l P l have been characterized biochemically, while the putative REV3 protein remains a “computer polymerase” (19). On the basis of conserved primary structure, biochemical activities, and subunit structure, yeast DNA polymerases I and I11 are the homologs of metazoan DNA polymerases (Y and 6, respectively (6, 7,20-25). POLX, a putative homolog of the mammalian DNA-polymerase-P gene, has been discovered in the nucleotide sequence of yeast chromosome 111 (26), suggesting that this polymerase is present in yeast. POLX could encode a protein of about 68 kDa. K. Shimizu and A. Sugino (unpublished observations) have partially purified from yeast extracts a DNA polymerase, tentatively called DNA polymerase IV, with properties characteristic of DNA polymerase @: it is a basic protein inhibited by 2’,3’-dideoxythymidine TABLE I

EUKARYOTIC DNA Yeast DNA polymerase gene

POLl (CDC17) POL2 POL3 (CDC2) POLX REV3 MIPI

Yeast DNA polymerase activity

Genetic map position<‘

14L 14L 4L 3R 16L

-

POLYMEHASES

I

a

I1

E

111 IV (?)”

6

Unknown M itochondrial

From 26 and 116; mapping of POL2 is described i n Table 11.

’, Identification of yeast DNA polviiierase IV as the product of POLX

eiikaryotic DNA polymerase f3 is pro\~isiwial(see text)

Corresponding higher eukaryotic activity

P

(?)‘I

Unknown

Y and thc homolog of higher

YEAST

DNA POLYMERASE

95

5‘-triphosphate, but not by N2-(4-butylphenyl)-2’-deoxyguanosine5’-triphosphate or aphidicolin, and requires a high Mg2+ concentration. The activity is intrinsic to a polypeptide of approximately 68 kDa. I t does not react with antibodies against DNA polymerase I, 11, or 111; whether it is encoded by the POLX gene is under investigation. A mammalian PCNA-independent (PCNA: proliferating -cell -nuclear antigen) DNA polymerase formerly conflated wi& 6 has been recognizedas being structurally distinct, and has been named E ( 1 1 , 27, 28). DNA polymerase E has been proposed (25, 27) to be the counterpart of yeast DNA polymerase 11, and resembles it by these criteria: the catalytic subunits of both yeast DNA polymerase I1 and human E polymerase are of very high molecular weight (>ZOO kDa) and, in contrast to the 6 polymerases, are active on a poly(dA).oligo(dT) template-primer in the absence of PCNA. Definitive evidence that the catalytic subunits of DNA polymerases I1 and E are in fact structural homologs has now been obtained from the primary structures of cDNA clones representing the human DNA polymerase E catalytic polypeptide (T. Kesti, H . Frantti and J. Syvaoja, personal communication). A unified nomenclature in which the yeast DNA polymerases were renamed according to their proposed metazoan counterparts was proposed in 1990 (25)even though it had not then been proven that DNA polymerases I1 and E are structural homologs. A standard nomenclature might be desirable, but the one promulgated disrupts the logic of the yeast system where the polymerases and their genes are given the equivalent Roman and Arabic numerals, respectively.

B. Class-B DNA Polymerases While all DNA polymerases may ultimately be related, they have been grouped into several classes on the basis of amino-acid sequence similarity (29). Class B contains the structurally related aphidicolin-sensitive DNA polymerases, and includes yeast DNA polymerase I1 as well as the metazoan ct polymerases. These related polymerases have been referred to as “a-like” (30),an imprecise term to convey primary structural homology, and confusing because class B also includes the metazoan 6 and E polymerases [it excludes the p and y enzymes; DNA polymerase p is related to deoxynucleotidyltransferase (31) and yeast DNA polymerase y is related to Escherichia coli DNA polymerase I, the archetypal class-A DNA polymerase (32, 33)].Furthermore, the a polymerases are atypical of class-B DNA polymerases in that they lack an intrinsic 3’+5‘ exonuclease (34, 35). Instead of “a-like,” we advocate use of the term “class B” (29),or the descriptive term “aphidicolin-sensitive. ” The primary structures of class-B polymerases are characterized by a

96

ALAN MORRISON AND AKIO SUGINO

series of conserved regions that occur in the same linear order in all members (20, 36). N o common system of labeling these regions has been universally accepted. We advocate using the numbering system of Wang et al. (36), also adopted by others (37, 38), in which the regions are given Roman numerals I-VI, in decreasing order of degree of conservation. This system must be modified by the addition of three conserved motifs of the 3’+5’ exonuclease active site, called Exo I, Exo 11, and Exo 111 (see Section IV, C). The reference to the left part of Region IV as Exo I’ by some authors (39-41) reflects the now-discredited theory that Exo I is located there (see Section IV, C), and is no longer useful.

II. DNA Polymerase II

Structure and Activities

A. Purification of Yeast DNA Polymerase II 1. EARLYDETECTION Yeast DNA polymerase I1 was first reported in the early 1970’s as a polymerase activity in mitochondria-free cell extracts chromatographically distinct from DNA polymerase I (42) and associated with an exonuclease activity (43).In more detailed studies, partially purified DNA polymerase I1 was shown to be antigenically and enzymatically distinct from DNA polymerase I: it required a deoxynucleotide template and primer, was most active with poly(dA).oligo(dT), and copurified with a 3’-+5’ exonuclease of similar heat stability, capable of excising a 3’-terminal mismatched nucleotide (44-46). In these and in a more recent corroborating report (47),DNA polymerase I1 appeared to be a polypeptide of 150-170 kDa. Following the discovery of the chromatographically and antigenically distinct DNA polymerase 111 (48,49), genetic evidence strongly implied that there are at least three DNA polymerases (in addition to the mitochondria1 enzyme) in yeast, since DNA polymerase I1 was still detected in mutants either of CDC2 (POL3) or C D C I 7 (POLI)(8, 47).

2. DNA

POLYMERASE

11 HOLOENZYA4E

Until the work of Hamatake, DNA polymerase I1 had been purified as a proteolyzed single polypeptide of about 150 kDa. Hamatake et al. purified to near-homogeneity a catalytically active single polypeptide of about 145 kDa but, in addition, observed a much larger catalytically active DNA polyinerase I1 polypeptide of 2200 kDa that copurified through five chromatographic steps and during glycerol gradient centrifugation with proteins of 80, 34, 30, and 29 kDa (50). This complex termed DNA polymerase I1*,

YEAST

DNA POLYMERASE

97

apparently constitutes the holoenzyme. Essentially no difference in enzyme activity was observed between the two forms of purified DNA polymerase 11: both were catalytically active in polymerase and 3'45' exonuclease assays and displayed highly processive polymerization utilizing poly(dA).oligo(dT) as template-primer. The 145-kDa and 22OO-kDa species were coantigenic and generated common products on partial digestion by Staphylococcus uureus V8 or Arg-C proteases. The 145-kDa species was evidently derived from the 2200-kDa species by proteolysis, concomitant with dissociation of the other polypeptides. Dissociation, or lack of formation, of the holoenzyme form was also observed in an extract from the po12-1 mutant, which yielded a genetically truncated polypeptide of about 131 kDa (51). The subunit structure of the holoenzyme is summarized in Table 11. We next describe cloning of the genes encoding the ?200-, 80-, 34- and 30-kDa species. The as-yet-unnamed gene encoding the 29-kDa putative subunit D has not been cloned and there is little information regarding it. Data on the po12-1 mutant and other mutants of DNA polymerase I1 and its subunits are summarized in Table 111.

B. Subunits of DNA Polymerase II 1. CATALYTIC SUBUNITA ENCODEDBY POL2 Definitive proof that DNA polymerase 11 is a unique activity came with the cloning of the POL2 gene encoding the catalytic subunit A of DNA polymerase 11: POL2 has a unique primary structure and genetic map position (51). The predicted POL2 protein is about 256 kDa, and displays sequence-similarity to class-B DNA polymerases in its N-terminal half. Evidence that POL2 encodes DNA polymerase I1 rests on three results: antibody to purified DNA polymerase I1 also reacts with fragments of POL2 protein expressed in E . coli; a hexapeptide terminal sequence of a Lys-C proteolytic fragment of DNA polymerase I1 catalytic polypeptide is exactly matched by a sequence encoded by the POL2 open reading frame; and a truncated DNA polymerase I1 polypeptide of the expected 131 kDa was partially purified from the poZ2-1 mutant disrupted in the middle of the POL2 coding region (but downstream from the polymerase domain). 2. SUBUNITB The 80-kDa polypeptide was designated subunit B. It copurified with the catalytic subunit A, and the two polypeptides appeared to maintain a 1:l stoichiometry in purified preparations (SO). The gene encoding the 80-kDa polypeptide was termed DPB2 (DNA polymerase subunit 2). Its sequence predicted a polypeptide of about 79.5kDa and showed no significant similarity to any protein sequence in the database (52).The 80-kDa polypeptide

S

TABLE I1 SUBUNITSTHUCTUHE OF DNA POLYMEMSE11" Proposed subunit

a 0s

Polypeptide

M,

X

lo-''

Open reading frame (codons)

Predicted protein ( M , )

Transcript

(W

Activities

14L, within 3.9 cM rad50" 16c 213, 30 c M dis-

2222

255,649

7.5

698 201

79,461 23,005

0.9

DNA polymerase, 3 '+ 5' exon uclease ? ATP binding

-

-

-

-

-

Gene

Map position

1

POI2 DPB2 DPB3

Stoichiometry

A

>200

c, C'

B

80 34, 30

1 =4

D

29

=4

tal his7

-

2.5

Data A r c from 50-54. To map POL2, a diploid isolated by crossing strain C<;954 (MATQ ura3 lys2 ho::LYSS ru&OA) with the pol2-1 derivativc of AMY50-IB (MATol lysl-l h i s - 7 a&-l hom-10 len2-1 rrra.3-52) WAS sporiilated. rad7OA was mnnitored b y srncitivity to mrthy ~ n c t h a r i c s ~ ~ l l ~ n and a t ep, d 2 - 1 b y thr Ura phcwutypc. Twelve tctradb giving Ciur vtal)lr spore$ all were of the parental ditype combination (unpublished observations). c H. Araki and A. Sugino (unpublished observations). '1 '1

+

TABLE 111 MUTANTSOF GENESENCODINGDNA POLYMERASE 11 Gene POL2

Allele

DPB2

DPB3

11 c

Amino-acid sequence change

A,

n, AND ~a Phenotype

Biochemical effect

DO12-1

URA3 insertion at base 3991 (BglII site)

Disruption at a.a. 1134

Truncated subunit A polypeptide; dissociation of subunits B and C

POL-2

URA3 insertion at base 1111 (RgZII site) LEU2 transplacement of bases 1315-7051 (EcoRV sites)

Disruption at a.a. 175 Deletion of a.a.’s 2422154 Asp-290+Ah, Glu-292-tAla

n.d.1)

Slow cell growth, increased spontaneous mutation rate Lethal

n.d.

Lethal

3’+5’ exonuclease reduced at least 100-fold

~012-3

(D W

Nuclentide sequence change

SUBUNITS

~012-4

A1460+ C. A1466- C

po1.2-3991 ~012-9‘

LEU2 insertion at base 3991 (RglII site) 625234A

~01.2-18c

C2719+T

dpb2A

LEU2 insertion at base 526

dpb2-1

n.d.

dpb3A

URA3 transplacement of 0.54-kb EcoRI-Hind111 region starting at base 346

Disruption at a.a. 1134 Met-644+Ile (within Region 11) Pro-7lO-tSer

n.d

Spontaneoirs mutation rate increased about 10- to 20-fold As for ~ 0 1 2 - 1

ts DNA polymerase I1 activity

t s cell growth

t s DNA polymerase I1 activity

t s cell growth

Disruption at a.a. 86 n.d.

n.d.

Lethal

Disappearance of subunit B from DNA polymerase I1 complex

ts

Deletion of a.a.’s 62-201

Altered chromatographic elution position and disappearance of subunits C and C ’ from DNA polymerase complex . .

Cell growth normal, increased spontaneous mutation rate

Data are from 35 and 51-54. n.d., Not determined. Note that nucleotide numbers fnr the pul2-9 and ~012-18motatioiis use the system of 51 and differ from those given in 53.

cell growth

100

ALAN MORKISON AND AKIO S U G I N O

was absent from D N A polymerase I1 holoenzyine partially purified from cells expressing the dpb2-I mutation, while subunits A and C were still detected (subunit D did not react with the mouse antiserum against D N A polymerase I1 used in the Western blots). The POL2 and DPB2 genes appeared to share a common cell cycle regulation (see below), and their temperature-sensitive and/or disruption mutants showed the same phenotypic consequences: cessation of genomic D N A synthesis; arrest of the cell cycle at the dumbbell morphological stage; and cell death (51-53). Our (unpublished) experiments provide genetic evidence for an interaction between subunits A and B in vivo. The po12-1 mutation confers an approximate halving of cell growth rate, resulting in small colony size (51). Thus, for cells grown in rich medium at 30"C, strain BJ3501 (54) and its pol2-1 derivative displayed doubling times of 103 1 and 208 8 minutes, respectively (mean and range of two determinations each). Since the pol2-1 mutation destabilizes the interaction between the catalytic subunit A and the essential subunit B in uitro (51), we tested whether overexpression of subunit B by increasing the DPB2 gene copy could suppress the po12-1 slowgrowth phenotype. Instead of po12-1, we used the ~012-3991allele, which is identical to po12-1 except that LEU2 instead of URA3 disrupts the POL2 coding region at nucleotide 3991 (Table 111). The inclusion of DPB2 on a multicopy plasmid substantially, but not entirely, suppressed the growth defect of po12-3991 (Table IV). The DPB2 plasmid had no effect on the growth of the parent strain, as judged by colony size.

*

*

3. SUBUNITC

The 34- and 30-kDa polypeptides are encoded by a single gene, DPB3. As with the POL2 and DPB2 genes, mouse antiserum against D N A polyTABLE IV DPBP GENECOPY NUMBER ~012-3991 S L O W G R O W T H P H E N O T Y P E "

E F F E C T OF INCREASED ON T H E

Relevant genotype

Doubling time (inin)"

Relative doubling time

POL2+ [YCP500]' td2-3991 [ Y C P ~ O ] ~012-3991 IYEpDPB21"

126 i 1 (2) 253 t 13 (2) 170 5 8 (4)

1 2.0

1.3

Derivatives of parent strain XSR03-2C (MATa hisl-7 [e112-3,-112 / ~ i i D - l ( i icm3-52 c a d ) were grown at 30°C i n synthetic medium lacking uracil to maiiitain the presence of plasmids. (7 Doubling times are expresaed as mean % i-ange or standard dcviatioii (nuinlter of determinations). a different isolate was uscd for each determination. Plasrnid YCp50 was used as the control lIHA3 plasmid. Plasrnid YEpDPB2 contains a KpizI-A'sil DNA fragmeirt containing the DPB2 gene (52) inserted into the K p n I - P r t l sites of the U M . 3 vector YEplacl95 ( l J 7 ) .

YEAST

DNA POLYMERASE

101

merase 11 holoenzyme was used to clone DPB3 from a h g t l l expression library (54). Antibody affinity-purified by reaction with the cloned DPB3 protein reacted with both the 34- and 30-kDa polypeptides by Western blotting, but not with the >200- or 80-kDa proteins. Furthermore, the dpb3A deletion mutant, which grew normally, yielded a DNA polymerase IT, partially purified through Mono-S column chromatography, consisting only of subunits A and B, lacking both the 34- and 30-kDa polypeptides. The 29-kDa polypeptide was present in the Mono-S column fractions but did not clearly co-chromatograph with subunits A and B, presumably because it had dissociated. (For this Western blot experiment, a rabbit antiserum that recognized all four subunits, A, B, C, and D, was used.) Since the DPB3 protein predicted from the sequence is only 23 kDa, the 34- and 30-kDa polypeptides are presumably either posttranslationally modified forms, or migrate anomalously in SDS-polyacrylaniide gels. The DPB3 protein has been expressed in insect cells using the baculovirus system (P. Ropp and A. Sugino, unpublished observations) where a 34-kDa, sometimes with a minor amount of 30-kDa, protein was produced. The 34- and 30-kDa polypeptides are denoted as subunits C and C'. The predicted DPB3 protein sequence contains two noticeable features: a region of 35 residues (residues 120-154) containing 63% acidic residues (primarily glutamate), including a run of 14 residues that contains 11 glutamates and one aspartate; and a possible nucleoside triphosphate-binding consensus sequence (55) in the N-terminal half of the predicted sequence (54). DPB3 protein expressed using the baculovirus system and purified to about 50% homogeneity bound ATP but not, to any significant extent, GTP, CTP, or 'ITP, as determined by SDS-polyacrylamide gel electrophoresis following challenge with 32P-labeled triphosphate and fixation using UV light (P. Ropp and A. Sugino, unpublished observations). Neither the partially purified DPB3 protein nor DNA polymerase I1 holoenzyme had detectable ATPase activity. Deletion of the DPB3 gene had no appreciable effect on cell growth (54). However, a modest (approximately 2- to 20-fold) increase in spontaneous mutation rate was measured by reversion assays in dpb3A strains, indicating that chromosomal DNA sequences are maintained less accurately when DPB3 protein is absent.

C. Stirnulatory Factors 1. S F I A factor called S F I that stimulates the activity of purified DNA polymerase I1 was partially purified (47). The stimulation occurred with poly(dA).oligo(dT), but not with activated DNA as template-primer and not with DNA polymerase I. SF I was subsequently purified as an apparent

102

ALAN MORRISON AND AKIO SUGINO

complex of polypeptides of 66, 37, and 13.5 kDa, with single-strand DNA binding activity intrinsic to the 66-kDa polypeptide (56).Although strikingly similar in subunit composition to yeast and human replication-factor A (57), the 66-kDa SF-I polypeptide was antigenically distinct from the 69-kDa DNA-binding polypeptide of yeast replication-factor A (56-58). Further investigation showed that the 66- and 37-kDa polypeptides are identical to the mitochondria1 heat-shock protein encoded by HSP6O and the yeast translation-initiation factor 4A, respectively (59). It is unclear whether SF I has any relevance to in uivo DNA replication. A second stimulatory factor, S F 11, was also found (47), but further reports on its identity or mechanism have not appeared. 2. PCNA

AND

RF-C

Although independence from PCNA is one of the criteria distinguishing the E from the 6 polymerases, there are several reports of an effect of PCNA on DNA polymerase 11. Hamatake et al. (50)found that PCNA increased the processivity of both the DNA polymerase I1 holoenzyme and the 145-kDa form with poly(dA).oligo(dT) as template-primer, while DNA polymerase I was unaffected. Lee et al. (60) found that, using singly primed coliphage M13 single-stranded DNA as primer-template in the presence of 0.13-M NaCI, synthesis by yeast DNA polymerase I1 (E) required hoth human PCNA and activator I (i.e., RF-C, replication-factor C), and was stimulated by E . coli SSB (single-stranded DNA-binding protein); in the absence of NaCI, synthesis required only SSB. With (dA),,o,).(dT)l,,,, in the presence of salt, but not in its absence, yeast DNA polymerase I1 required PCNA, human SSB, and human RF-C (60). Similar data were reported using yeast PCNA and RF-C (61, 62). These results do not necessarily imply any direct interaction with DNA polymerase 11. Perhaps SSB stimulates DNA polymerase I1 non-specifically by smoothing out regions of secondary structure and reducing nonproductive binding of the polymerase to single-stranded DNA, while RF-C and PCNA may bind the primer-template and present the primer terminus to any of a variety of DNA polymerases. On the other hand, it is possible that some of these stimulations do reflect protein-protein interactions involving DNA polymerase 11. We speculate that PCNA and RF-C, while specific for DNA polymerase 6, might have cellular hoinologs specific for DNA polymerase E, and that some functional cross-reactivity can occur.

D. Mammalian DNA Polymerase E Purified HeLa cell DNA polymerase E appears to have a subunit structure different from that of yeast DNA polymerase 11. It has been purified as a complex between a >200-kDa catalytic subunit and a 55-kDa subunit (27,

YEAST

DNA POLYMERASE

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60, 63, 64). The >200-kDa subunit was cleaved by trypsin into 136- and 122kDa polypeptides, the smaller polypeptide containing the polymerase and, probably, 3'+5' exonuclease activities (64).This is reminiscent of the ~ 1 5 0 kDa polypeptide of yeast DNA polymerase 11, which is presumably derived by endogenous proteolysis from the 2200-kDa polypeptide. Purified calfthymus DNA polymerase E appears to be composed of either a single 140kDa catalytically active polypeptide (64),or polypeptides of 140, 125, and 40 kDa (28), the 140- and 125-kDa polypeptides being catalytically active (65). The apparently different subunit compositions of the yeast and mammalian E polymerases might reflect different physiological states of the enzymes rather than species differences (S. Linn, personal communication). If the mammalian and yeast E DNA polymerase subunit structures have diverged, it might suggest a divergence in roles of the polymerases. Alternatively, it is possible that the 55- and 80-kDa subunits of the yeast and human E DNA polymerases are homologs, though differing in size; other polypeptides corresponding to subunits B and C of yeast DNA polymerase I1 might simply have dissociated from the mammalian polymerase during purification. Siegal et al. (66) observed a functional interaction between calf-thymus DNA polymerase E and a 5'-t3' exonuclease similar to enzymes found in HeLa cells (67) and mouse cells (68). The exonuclease degraded a singlestranded DNA or HNA primer annealed to a template. When two primers, separated by a three-nucleotide gap, were annealed to an M13mp18 DNA template, degradation of the downstream primer in the presence of DNA polymerase E was dependent on provision of dNTP's and synthesis from the upstream primer; conversely, DNA polymerase a or 6 did not prevent degradation of the downstream primer. Siegal et al. (66) suggested a model in which the E polymerase is involved in replicating the lagging strand.

111. Cell Cycle Regulation In S . cerevisiue, at least 15 genes encoding proteins involved in DNA replication are coordinately expressed at the G,/S-phase boundary of the cell cycle (69). This expression requires an upstream cis-acting nucleotide sequence motif exemplified by the MluI restriction enzyme recognition sequence. The FOL2, DPB2, and DPB3 genes appear to belong to this set of genes: all contain at least one occurrence of the MZuI motif, or a variant, in their 5'-untranslated regions, and their transcripts were expressed at the G,/S-phase boundary (51-54). Periodic expression of the POL2, DPB2, and DPB3 genes was demonstrated with yeast cells synchronized using the a mating pheromone, a feed-starve procedure, or elutriation. This coregula-

104

ALAN MORRISON AND AKIO SUGINO

tion argues against the possibility that the DPB2 and DPB3 proteins copurify fortuitously with D N A polymerase 11, and provides circumstantial evidence that D N A polymerase I1 acts during S-phase.

IV. Domain Structure of Catalytic Subunit A. Polymerase Domain Class B polymerases are characterized by a series of conserved regions that occur in the same linear order in all members (20, 36). The extent of these conserved regions presumably defines the enzyme activity domain(s). Mutations conferring altered sensitivity to nucleotide analogs have been mapped to positions in and around Regions 11-V (38, 70, 71),and mutation of specific residues in Regions I and XI eliminated D N A polymerase activity (72-77). Inspection of the predicted amino-acid sequence of the aphidicolinsensitive D N A polymerase I1 showed that it belongs to class B, and contains the conserved regions I-VI in the same linear order identified by Wang and others (51, 36). Figure 1 shows a comparison of the domain structures of D N A polymerase I1 and the other aphidicolin-sensitive D N A polymerases of yeast (including the predicted REV3 polymerase). Two additional conserved regions are apparent, numbered VII and VIII (Fig. 1). Region VII contains several basic residues and a tyrosine, and is conserved in many class-B D N A

II

IV

POL1 REV3

I

vi

n1

I

vn v

vni

QS

I -

3

POL3

POL.? Ex01 EXO

n

Exolll

FIG 1 Conserved regions of yeast DNA polymerase5 Yeast DNA polymerase5 I, 11, and 111, and REV3 protein, identlfied by their gene svmbols, are schemati7ed d S horiLorrta1 line5 with their N-termini to the left and C-termini to the right Boxes represent conserved region\ Solid boxes labeled I-VI represent conserved reglons defined by Wang et a1 (36) Sold boxes VII and VIII are two additional con5erved reg10115 Hatched boxes labeled C \ s reprewnt cysteine-rich region\ Open boxes (below lines) labeled Exo I, Exo 11, a i d Exo I11 represent coiiserved motifs containing 3’+5’ exonudea5e active-site residues (35), Exo I1 is coincldent with the right part of Region IV Region VII was dexribed for herpes-simplex-\.irrr5 DNA polvmerase (39, and is the same as Region 9 of 81, region IV of 118, and Region H of 119 It is characterized by the Ly\-Lys-Arg-Tyr motif ( r e s h e \ 96G-969 of yeast DNA polvmeIase 11) Region VIII 15 characterized by the Asp-X-X-Tyr-Tvr motif (residues 1142-1146 Of yeast DNA polymerase 11, X IS a non-conwrved amino acid)

YEAST

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POLYMERASE

105

polymerases, including herpes-simplex-virus DNA polymerase (38). Region VIII is characterized by the Asp-X-X-Tyr-Tyr motif (X is a non-conserved amino acid), and is not clearly conserved in class-B polymerases other than those shown in Fig. 1 and their honiologs. The N-terminal half of DNA polymerase 11, excluding Region VIII, contains the polymerase and 3’+5‘ exonuclease activities, since a truncated DNA polymerase I1 polypeptide with these activities was partially purified from po12-1 mutant cells (51; A. Sugino, unpublished observations).

B. C-Terminal Half The amino-acid sequence of DNA polymerase I1 contains a C-terminal cysteine-rich region, thought to comprise a zinc-finger DNA-binding domain. Similar regions occur at the C-termini of metazoan ci and 6 polymerases and their yeast counterparts, and in the predicted REV3 polymerase, but are absent from other aphidicolin-sensitive polymerases. DNA polymerase 11 differs strikingly from other class-B enzymes in possessing a large unique C-proximal region. Perhaps even more strikingly, neither this unique region nor the Cys-rich region nor Region VIII is required for DNA polymerase II function either in uitro or in uiuo, as determined from analysis of the po12-1 mutant (51).Since the partially purified poZ2-1 mutant protein lacked associated subunits, the C-terminal half of the catalytic subunit is apparently required for maintenance of the holoenzyme form. The partial suppression of the po12-1 slow-growth phenotype by increased DPB2 gene copy-number (Table IV) argues that a significant function of the C-terminal half is to hold subunit B at the site of action of DNA polymerase 11, where it has an essential role.

C. 3’+5’ Exonuclease Active Site 1. CONSEWED

ACTIVE SITE

Five 3’+5’ exonuclease active-site residues have been assigned in DNA polymerase II on the basis of amino-acid sequence alignments with other class-B D N A polymerases and with E . coli D N A polymerase I (35).The 3’+5’ exonuclease active site of the Klenow fragment of E . coli DNA polymerase I has been exquisitely detailed by crystallographic and mutational studies (78-80). Several groups proposed that amino-acid sequence motifs corresponding to sequences in the Klenow 3’+5’ exonuclease active site could be recognized in aphidicolin-sensitive DNA polymerases (71, 81, 82). The most comprehensive statement of this hypothesis was made in a seminal paper by Salas and co-workers, who proposed that the 3’+5’ exonuclease active site residues are embedded in three amino-proximal sequence motifs, Exo I, Exo 11, and Exo 111 (30). While fundamentally correct, this report contained some sequence alignments that were subsequently challenged (9,

EX0 111

* S.cerevisiae

.

S pombe

Pol 11 Pol I1 Pol 111 PO1 6 Pol 6 PO1 6

S.cerevisiae S .pombe Human Bovine P.falciparum pol 6 Herpes simplex virus Varicella-zoster Epstein-Barr virus Cytomegalovirus Baculovirus Vaccinia virus Fowl poxivirus Adenovirus-5 Bacteriophage T4 E. coli P o l I1 Chlorella virus C.biennis virus Maize mitochondria S1 N.intermedia plasmid K. lactis pGLK-2 Bacteriophage PRDl Bacteriophage M2 Bacteriophaqe 629 S.cerevisiae Pol 1 S.pombe Pol a Human Pol a D.melanogaster P o l a S.cerevisiae Rev3

286-V M A 265-v M A 317-1 M S 296-1 M S 312-V L S 311-V L S 303-1 L S 364-L M C 345-L L C 292-A L A 297-C L S 192-L S C 137-Y L F 160-Y L L 137-F V T 108-V A N 152-W V S 186-1 A S 163-8 V S 221-F F V 262-1 M T 363-E V F 13-1 A A 5-M F S E-M Y s

F F F F F F F F F F F Y L F Y C I

a I D I D I D I D I D I D I D I D I D I D I D I D I D I D V D I D I D I D I D L D L D I D F

*

O E E E E E E E E E E E E E E E E W E I E A E L E F E F E C D F E C ~

* T T K-295 T T K-274 C A G-326 C A G-305 C A G-321 C A G-320 C I K-312 C K A-373 C K S-354 C L G-301 C M s-306 T H S-201 C H F-146 C Q F-169 T Y "-146 V T G-117 T T R-161 T Y S-195 C Q H-172 T L L-230 T R s-271 S F s-372 T D P-22 T T T-14 F T ~T-17 T

374-1 S 353-1 V 398-1 I 377-L I 393-1 T 392-1 T 386-L T 462-V T 443-A T 375-V T 404-V T 276-1 L 234-V V 247-V I 271-1 V 210-F T 220-1 I 268-5 I 263-1 L 306-V Y 336-V Y 416-L Y 67-1 Y 55-L Y 58-L Y 635-1 I 621-Y F 634-1 V 660-1 V 764-L S

T T G G G G G G G G G D

T T G G G G T F T A A F F G G G T

F Y Y Y Y Y Y Y Y Y Y F F E H

N N N N N N N N N N N

G G T I I I I I I V I N G N G

N N W N W N Y Y H H W H

N

N N N

G

I I V L G L F G G L L L F

Y N H N H N H R H D H N I F D S G F E I

D D T C Q Q

F F N N

*

F D W P F I H N R-393 F D W P F V D A R-369

F H I P Y L L N R-414

F D I P Y L L D R-393 N F D L P Y L I S R-409 N F D L P Y L I S R-408

I N I N V N A N N S D V H N N N N G E G V Q W Q H S S Q S Y S G G K . K .K Q N E . Y G M D H N

F D L F D W F D W F D W F D L F D L F D L F D I F D E F D V F D L Y D L F D F F D G F D G Y D Y F D F F D G F m G V Y L M C Y F E L C Q L F S W

P Y P F A F P Y K Y P Y R Y

R Y I V P Y R M R Y T Y I M I F Q H L F A F A F D V S E N G

V V V Y

I L I I I I I I L I L I I I I V L I I L L L I I

L L M L L L T

S A M Q

N R-402 A K-478 E K-459 D R-391 T R-420 G R-292 N R-250 G R-263 A Q-287 N R-226 K H-236 G R-284 G R-279 S F-322

H Q L I D 1-352 L P Y-432 M K Y-83 V N W-70 I N W-73 A H R-651 L S R-636 L Q R-650 T D Q-676 I E R-780

470-L 449-L 513-L 492-L 509-L 507-L 501-1 574-1

555-1

490-L 535-V 386-1

430-M 441-M

435-T 317-Y 328-L 390-1 464-1

406-S

470-L 546-A 142-1

159-E 162-E

*

S V S D A V A-480 A Q Y S V S D A V A-459 S E Y

A A A A A G G G G A A E L I A A A L I I L Y Y

V I V V T E E M R K

R R D

S T A Y T K E E E A

Y C L K D Y C L K D Y C L K D Y C L K D Y C I K D Y C I Q D Y C I Q D Y C V Q D Y C L Q D Y N V Q D Y c I n D Y C I H D Y C A L D Y N I IH Y N L K D Y A R K D Y C T H D Y L K Q D Y C E I D Y C K V D Y L K G D Y I K N D m 1 K N m

A A A A G

S S S A C

A A V V C T T

I

T V C I I

Y L-523 Y L-502 Y L-518 F L-517 V L-511 L L-584 A L-565 A L-500 V L-545 M L-396

c

C Q E E D V L M L V E Q

L-440

L-451 V-445 5-32? L-738 L-400 L-474 1-416 A-480 A-556 T-152 1-169 1-172

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35, 83, 84) and that apparently led to two misconceptions: first, Exo 1 was mislocated in the left part of Region IV instead of proximal to it (35, 39, 40); and second, the idea that these active-site residues occur in all aphidicolinsensitive DNA polymerases (35).A modified version of the basic hypothesis of Salas and co-workers is given in Figs. 1 and 2. Exo I is located upstream from Region IV, Exo 11 is coincident with the right part of Region IV, and Exo 111 is between Regions IV and 11.

2 . MUTATIONOF ACTIVE-SITERESIDUES Identification of the Exo motifs has been tested by site-directed mutagenesis of specific residues in several polymerases. The effects of these mutations have been measured by assay of the 3'+5' exonuclease and polymerase activities of the partially or completely purified protein, and/or by genetic assay of a spontaneous mutator phenotype, which is the expected phenotypic effect of a loss of exonucleolytic proofreading. The identification of the conserved Asp-Ile-Glu motif (D-I-E in the single-letter code) upstream from Region IV as Exo I is supported by the results of site-directed mutagenesis of the proposed Asp and Glu active-site residues in yeast DNA polymerases 111 and I1 (35, 39). In the case of DNA polymerase 11, mutation of both the Asp-290 and Glu-292 residues to alanine reduced the ratio of 3'+5' exonuclease and polymerase activities at least 200-fold. With DNA polymerase 111, mutation of the corresponding residues increased the spontaneous mutation rate more than 100-fold. Exo TI, containing the conserved Asp residue corresponding to Asp424 of the E . coZi DNA polymerase I Klenow fragment, lies in the right part of Region IV. Mutation of the corresponding Asp residue in yeast DNA polymerase 111 and in bacteriophage +29 DNA polymerase specifically reduced 3'+5' exonuclease activity (30, 39). Experimental support for the identity of the Exo 111 motif came from the 3l-5' exonuclease-deficient Asp-32bGly mutation of coliphage T4 DNA FIG. 2. Alignments of aphidicolin-sensitive DNA polymerase amino-acid sequences proposed to contain conserved 3'-95' exoiiuclease active-site residues. (The one-letter code is used.) Numbers refer to amino-acid residues. The three conserved regions Exo I, Exo 11, and Exo 111 (see Fig. 1)are shown. Asterisks mark invariant residues proposed to correspond to the following 3'-+5' exonuclease active-site residues of the Klenow fragment: in order (from left to right) D355, E357, D424, Y497, and D501. Boxed are residues whose mutation led to a decreased ration of 3'+5' exonuclease and DNA polymerase activities. DNA polymerases known to possess 3'+5' exonuclease activity appear above the horizontal line; below the horizontal line are a DNA polymerases and the predicted REV3 protein. Abbreviation: Pol, DNA polymerase. Sequences are from the Genbank/EMBL database. The S. pornbe DNA polymerase I1 sequence is the unpublished work of J. Sebastian and A. Sugino. S. cereoisiae DNA polymerase 111 is numbered according to 120.

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ALAN MORRISON AND AKIO SUGINO

polymerase, isolated using a genetic screen for isolates with high mutator activity (83).The interpretation is somewhat clouded because the Glu-lSl-+hla mutant rather than wild-type T4 DNA polymerase was used for the screen; however, the Glu-lSl--+Alamutation alone had no significant effect on 3'-+5' exonuclease activity, whereas the Glu-l91-+Ala, Asp-324-Gly double mutation reduced it about 100-fold. With $29 DNA polymerase, mutation of the conserved Exo I11 Tyr and Asp residues reduced the exonuc1ease:polymerase activity ratio: the Asp-169-Ala mutation reduced the ratio lO3-fold, and the T y r - 1 6 h P h e and T y r - 1 6 5 - K ~mutations ~ reduced it 13- and 24-fold, respectively, without diminishing polymerase activity as determined by filling-in of extended 5'-termini or by using (dC),;(dG),, as primer-template (41).

3.

REGION

IV

IN

a POLYMEKASES

The Exo I and 111 motifs, and the conserved Asp residue in Exo 11, can be located in all class-B polymerases except the a polymerases and the predicted REV3 protein (Fig. 2; 35). The a polymerases and the putative

REV3 protein are thus predicted, on the basis of sequence alignments, to lack an intrinsic 3'-+5' exonuclease activity. Although several different alignments have been presented to suggest conservation of the Exo motifs in a polymerases and the predicted REV3 protein (21, 30, 40, 41), in no case are the five predicted active-site residues absolutely conserved within a b'w e n a polymerase, nor is any of these residues invariant among the a-polymeraselREV3 subset. This contrasts with the other more-than-20 class-B polymerases in which these five residues are invariant. Consistent with this, the human a-polymerase catalytic subunit is devoid of 3'-+5' exonuclease activity (34). Earlier reports of 3'-+5' exonuclease activity associated with the a-polymerase catalytic subunit can be attributed to contamination and the then-reasonable belief that these essential replicases ought to possess a proofreading function (85, 86). We conclude that the 3'1.5' exonuclease is neither cryptic nor inactive, but is simply not present in the a-polymerase catalytic subunit. The above conclusion presents a paradox, since Region IV, which contains the Exo I1 motif, is conserved in ci polymerases. It appears that they possess at least a part of the 3'-+5' exonuclease activity domain, but lack the actual active-site residues. The Region-IV domain presumably has some function in addition to 3'-+5' exonuclease activity. We can speculate that this function might be related to binding single-stranded DNA. The crystal structure of an editing complex showed that the Klenow fragment bound four nucleotides of single-stranded DNA to the 3'-+5' active site (87).This complex is stabilized by hydrophylic interactions between the last three bases at the 3'-terminus and amino acids other than the four invariant acidic residues and tyrosine in the Exo I, 11, and 111motifs (87, 88). Consistent with the existence of a function other than 3'+5' exonuclease activity in Region IV,

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mutation of residues of herpes-simplex-virus DNA polymerase Region IV, mapping upstream from the conserved Asp residue in Exo 11, inactivated the polymerase (89). Similarly, the cdcl7-2 mutation, a Gly-637-Ala change in Region VI of S. cereuisiae DNA polymerase I, caused a temperaturesensitive phenotype indicating conditional inactivation of the protein (90). With T4 DNA polymerase, the Glu-l91+Ala, A s p - 3 2 b G l y double mutation appeared to reduce polymerase activity on a gapped template-primer about 30-fold, but only about %fold with a “nicked” template-primer (83). [Mutation of conserved acidic residues in the Exo I or I1 motifs did not detectably reduce DNA polymerase activity of the +29 enzyme, or of yeast DNA polymerase I1 or I11 (30, 35, 39).]Mutant $29 polymerases altered in the Exo I11 motif had a considerably reduced ability to replicate +29 DNA, though they were not defective in DNA synthesis assays in which strand displacement was not required, suggesting that the conserved Region IV domain might function in strand-displacement (41). Conservation of such a function in Region IV can explain why a polymerases retain this region. 4. ONLY6 AND E DNA POLYMERASES ARE PREDICTED TO POSSESSAN INTRINSIC3’+5’ EXONUCLEASE The amino-acid sequence alignments of the conserved 3’+5‘ exonuclease domain discussed above lead to the interesting proposition that, of the known nuclear DNA polymerases (i.e., excluding the mitochondria1 polymerase), only the 6 and E DNA polymerases possess an intrinsic 3‘+5’ exonuclease activity. The catalytic subunits of a polymerases, and the structurally related REV3 protein, lack the 3‘+5‘ exonuclease active-site residues. Yeast DNA polymerase I holoenzyme is devoid of 3‘+5’ exonucleolytic proofreading activity (91).DNA polymerase p lacks both the conserved 3’+5’ exonuclease domain (31)and an intrinsic 3’+5‘ exonuclease activity. Deoxyribonuclease V is sometimes associated with DNA polymerase p, but does not have the properties of a proofreading exonuclease (10).The 6 and E DNA polymerases therefore appear to be the only candidates with the intrinsic ability to perform exonucleolytic editing during DNA replication. While it is conceivable that an autonomous proofreading 3‘-+5‘ exonuclease exists, there is no precedent for this: the rule is that polymerase and 3’+5’ exonuclease coexist in the same polypeptide chain or, as with E . coli DNA polymerase I1 holoenzyme, as tightly complexed subunits, reflecting the mechanistic coupling of the two activities (17).

D. POL2 Homologs in Fission Yeast and Fruit Fly

Parts of genes homologous to POL2 have been amplified from genoinic DNA of Schizosaccharomyces pombe and cDNA o f Drosophila melanogaster using PCR and primers similar in nucleotide sequence to the DNA encoding

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Regions IV and I11 of S. cerevisiue DNA polymerase 11. A DNA fragment of approximately 800 base-pairs was amplified from D . melanogaster larval cDNA; its nucleotide sequence contained an open reading frame encoding a 272-amino-acid sequence that was 57% identical to the amino-acid sequence between Regions IV and I1 of S. cerevisiue DNA polymerase I1 (A. B. Clark and A. Sugino, unpublished observations). A similar DNA fragment was amplified from S. pombe DNA (J. Sebastian and A. Sugino, unpublished observations). A DNA clone containing part of the S. pomhe POL2 homolog was isolated using the PCR DNA fragment to probe a X library of S. pombe DNA. The DNA sequence of this clone showed part of an open reading frame corresponding to the N-terminal half of DNA polymerase 11, with all of the conserved regions shown in Fig. 1 present. Residues 192-1188 of S. cerevisiae DNA polymerase I1 showed 62% identity to the translated sequence of the S . pomhe clone.

V. Genetics of 3’+5’ Exonuclease A. Spontaneous Mutator Phenotype of 3’+5’ Exonuclease-Deficient Mutants Since the role of the 3’+5’ exonuclease is to remove incorrectly inserted nucleotides during polymerization, its inactivation is expected to lead to an increase in spontaneous single-base mutations in vivo, as is observed with mutants of dnaQ, which encodes the 3’--+5’exonuclease E subunit of E . coli DNA polymerase I11 (92).We created the exonuclease-deficient ~012-4and POD-01 strains by altering the Phe-Asp-Ile-Glu Exo I motifs to Phe-AlaIle-Ala (35, 93). Similar mutants of POL3 were constructed by Simon et al. (39). We measured mutation rates either by reversion assays or by forward mutation to 5-fluoroorotic acid resistance (94, 95) of a URAS reporter gene inserted near a defined replication origin (ARS306) on chromosome 111. The relative URA3 mutation rate for POD-01 (i. e., the mutations per cell division of the POD-01 strain divided by the mutations per cell division of the P O U + strain) was 130 (94, which compares to about 400 for mutation to canavanineresistance reported for similar pol3 mutants (39). The relative URA3 mutation rate for ~012-04was 12 (unpublished observations); this compares with 20, the average of the relative reversion rates for six different markers: his72 and ade2-1 (unpublished observations), and ade5-1, hisl-7, leu2-1, and h o d - 1 0 (35). A limited number of the mutations produced by each mutant polymerase were sequenced. Both ~012-4 and POD-01 generated exclusively singlebase changes within the URA3-coding region, but gave different mutational

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spectra. Twenty sequenced uru3 mutations from POD-01 occurred at 15 locations, and were predominantly transitions and single-base additions or deletions, with only one (G.C+T.A) transversion. Nineteen sequenced uru3 mutations from ~012-4occurred at nine locations and, except for a singlebase deletion, consisted of G.C+A.T, C.G+A.T, and T.A+A.T changes. Between both polymerase mutants, all classes of single-base mutation were represented in these spectra except G.C-42.G transversions.

B. Epistatic Relationships 1.

RELATIONSHIP BETWEEN

OF

3’+5’ EXONUCLEASES

DNA POLYMERASES I1 AND I11

While the ~012-4and POD-01 exonuclease-deficient mutants grew normally, the double-mutant POD-01 ~012-4haploid was inviable, as shown by tetrad analysis of spores from heterozygous diploids. The inferred pot%-01 pd2-4 spores formed microcolonies of inviable cells having no unique terminal morphology, consistent with death from a catastrophically high rate of unedited errors. We posited that such an error rate might be protected against by diploidy, and successfully constructed a viable double homoallelic pot%-01 /pot%-01 po12-4/po12-4 diploid. This diploid displayed a relative his7-2 reversion rate of about 2 x lo3, compared to 240 and 9 for the pot%-01 and po12-4 single-mutant haploids, respectively (unpublished observations). As with similar mutants constructed by Simon et al. (39),pot%-01 was partially dominant, still displaying a demonstrable mutator effect in the presence of POL3 (93).We transformed a plasmid carrying the pot%-01 allele into a haploid strain containing the wild-type POL3 gene, a genetic configuration denoted as POL3 [poD-O1]. We then compared the relative mutation rates of haploids of genotypes ~ 0 1 2 - 4 ,POL3 [pot%-Ol], and ~012-4POL3 [poD-OI]. We observed simple additivity between the relative URA3 mutation rates of POL3 [POD-011and ~012-4mutants, indicating that the epistatic relationship between the two exonucleases is that of competition rather than action in series (unpublished observations). The synergy observed in the pot%-01 ~012-4double mutant indicates that one 3’+5’ exonuclease can compensate for the absence of the other. These results suggest that there is no autonomous 3‘+5‘ exonuclease that can effectively substitute for those of DNA polymerases I1 and 111. Interestingly, the idea that the 3’+5‘ exonuclease of one DNA polymerase could compensate for the lack thereof in another came from a search for an exonuclease activity that would allow DNA polymerase a to elongate an annealed primer with a mismatched 3’-terminus (96). This search found an activity identified as DNA polymerase 6.

112

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&LATIONSHIP

ALAN MOHHISON AND AKIO SUGINO

WITH

MISMATCHk P A I H

In yeast, what appears to be a generally conserved mismatch correction system (97-99) requires PMSI, which is structurally related to the prokaryotic mutL and hexB (100). The P M S l system probably corrects mismatches arising during D N A replication. Thus, the occurrence of the MluI sequence motif in the 5'-untranslated region of PMSl, and the periodic expression of its transcript at the G,/S-phase boundary, suggest it is under the same cell cycle control as many D N A replication genes (93). Furthermore, a pmsl deletion mutant displayed a spontaneous mutator phenotype of about 31-fold (101). We observed that a ~ 0 1 2 - 4 pins1 double mutant was viable, but grew poorly, and had a relative URA3 mutation rate that was approximately the product of the relative mutation rates of the pol2-4 and pmsl single mutants (unpublished observations). This multiplicative relationship indicates that the POL2 3'-+5' exonuclease and P M S l act in series. We also observed multiplicity between the relative mutation rates of pols-01 and pmsl mutants, indicating that the POL3 3'+5' exonuclease and P M S l act in series (93). The results, then, are consistent with the idea that mismatches produced by either D N A polymerase I1 or 111 are normally corrected by the P M S l mismatch correction system (102).

VI. The DNA Repair Polymerase A. In Vitro Systems Human D N A polymerase E was identified as a factor required to reconstitute an undefined pathway of D N A repair synthesis in UV-irradiated permeabilized human diploid fibroblasts, thus implicating the E polymerase in this process (103). PCNA (and by implication, D N A polymerase 6) was required for nucleotide excision repair in a HeLa cell-free system (104).In nuclear extracts of human cells, D N A polymerase was identified as the polymerase responsible for short-patch D N A repair of processed apyrimidinic sites (105). In yeast, base excision repair synthesis of D N A either containing uracil or treated with UV or osmium tetroxide was examined in cell-free nuclear extracts (106; Z. Wang, X. Wu and E. C . Friedberg, personal communication). Repair synthesis did not occur in ~012" mutant extracts, and could be restored by addition of purified D N A polymerase 11. Repair synthesis was stimulated in extracts, while addition of purified D N A polymerase 111 was inhibitory. This suggests that D N A polymerase 111 competes with D N A polymerase I1 to sequester the primer-template, but that D N A polymerase

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111 performs little DNA synthesis, perhaps because it lacks a cofactor such as PCNA.

B. Genetic Evidence for Polymerases Involved in DNA Repair 1. DNA POLYMERASE I1 DNA polymerase I1 has been proffered as the “repair polymerase” (8, 47). Contrary to the implicit assumption, DNA repair is not a single process

but an array of different pathways that have been dissected at least partially by genetic studies (107). It cannot be presumed that a particular polymerase acts in repair but not in replication, or that a single polymerase performs all DNA repair, or that each repair pathway has a dedicated polymerase. If DNA polymerase I1 is involved in the repair of damaged DNA, it might be possible in principle to obtain radiation-sensitive mutants either in POL2, or in DPB2 or DPB3. However, none of the mutants (Table 111) confers increased sensitivity to UV, y-rays, or methyl methanesulfonate. Nor has any poZ2 allele been picked up in genetic screens for mutants defective in DNA repair. However, alleles of both POL1 and POU, but not of POL2, conferring sensitivity to methyl methanesulfonate were found in a genetic screen for mutants with a hyper-recombination phenotype (108). While not excluding the possibility that DNA polymerase I1 acts in the repair of damaged DNA, the fact that POL2 is an essential gene means that it cannot be required solely for any known DNA repair pathway, since all known DNA repair pathways are dispensable for cell growth (107, 109-111).

2. REV3,

THE

“COMPUTERPOLYMERASE”

One minor repair pathway, the REV3 pathway, probably does have a dedicated polymerase: the REV3 “computer polymerase” (19).This pathway, one of at least two repair pathways in the RADG epistasis group (the other is the RAD18 postreplication repair pathway), is minor in terms of overall repair, but is responsible for virtually all mutations induced by environmental DNA-damaging agents. Hypothetically, the pathway acts by translesion synthesis, i.e., by insertion ofa base opposite a position in the template that contains a damaged or aberrant base; the predicted lack of an intrinsic REV3 3‘+5‘ exonuclease (Fig. 2) is in accord with this.

3. DNA POLYMERASES I AND 111 The POL1 transcript accumulates following UV treatment of cells, suggesting a role for DNA polymerase I in the repair of UV damage (112). Yeast DNA polymerase I, however, appears not to be required for the repair of

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ALAN MORHISON AND AKIO SUGINO

X-ray-induced single-strand breaks, as determined by alkaline sucrosedensity-gradient sedimentation of radiolabeled DNA from pollt' cells (113). Conversely, DNA polymerase I11 was implicated in recombinational repair induced by UV or y-ray treatment of cells: in an ingeniously designed experiment, stationary-phase diploids heteroallelic for two different pol3f5 (cdc2) mutations were given sublethal irradiation doses and incubated in fresh medium at the non-permissive temperature ( I 14). In a control experiment with a diploid heteroallelic for cdc4, a cell-cycle gene not required for recombinational repair, DNA damage-induced recombination created wildtype CDC4 genes in some cells, which permitted growth and colony formation at a dose-dependent frequency. The pol3 heteroallelic diploid, however, failed to form colonies.

VII. Role of DNA Polymerase II in DNA Replication The involvement of DNA polymerases I and I11 or their higher eukaryotic a and 6 counterparts in replicating DNA is well documented (9, 10, 14, 15). Conversely, the REV3, M I P I , or POLX genes can be inactivated without causing cell inviability (or any growth defect in the cases of REV3 and POLX) and are thus not essential for genoinic DNA replication (19,115;P. Hopp and A. Sugino, unpublished observations). After the cloning of POL2, three lines of argument led to the suggestion that DNA polymerase I1 also participated in DNA replication. First, POL2 was essential for normal cell growth (51), whereas all known DNA repair systems are dispensable. Second, POL2, DPB2, and DPB3 appeared to be coregulated with DNA replication genes during the cell cycle (52-.54). Third, the terminal arrest morphology of yeast cells lacking POL2 or DPB2, or cells carrying temperature-sensitive p012 or dpb2 alleles held at the nonpermissive temperature, was typical of arrest during S-phase (51-53). The phenotypic characteristics of poi2 conditional mutants are indistinguishable from those of conditional mutants of pol1 or pol3. The finding that the nonessential UNGl (encoding uracil DNA glycosylase) and P M S l genes are also under the same cell-cycle control as DNA replication genes (93, 110) leaves open the possibility that POL2 (or, indeed, POL1 or P O U ) is required to repair a hypothetical form of lethal spontaneous damage that occurs during replication: this, by definition, would be an essential part of DNA replication and the argument thus becomes a tautology. Direct evidence for a replicative role of DNA polymerase I1 came when

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it was shown that incorporation of labeled precursor into D N A of pol2 or dpb2 temperature-sensitive mutant cells ceases after shift-up to the nonpermissive temperature (52, 53). Analysis by FACS (fluorescence-activated cell sorting) also showed that DNA synthesis in poZ2ts cells held at the nonpermissive temperature ceased with the same kinetics shown by poll t~ cells (53). More recently, Budd and Campbell ( 1 1 5 ~ )used alkaline sucrosedensity-gradient analysis of DNA synthesis products to show thct no chromosomal-sized DNA is synthesized after shift of an asynchronous p 0 l 2 ~ ~ culture to the non-permissive temperature. The DNA profiles of replication intermediates from the pol2ts mutants were similar to those observed with DNA synthesized in mutants deficient in DNA polymerase I under the same conditions. Further evidence implicating DNA polymerase I1 in DNA replication comes from the spontaneous mutator phenotype of the exonuclease-deficient ~012-4 mutant. While a spontaneous mutator phenotype may also arise in DNA repair mutants, the P O & - 4mutation does not confer sensitivity to DNA-damaging agents, and the epistatic relationships discussed above link the DNA polymerase I1 3 ' 4 5 ' exonuclease with DNA replication. We have defended a model of the DNA replication/error correction cycle in which DNA polymerases a, 8, and E copy the template DNA, the 6 and E 3'+5' exonucleases excise misincorporated nucleotides, and remaining errors are corrected by the serial action of the P M S l mismatch correction system (102). Several models have been proposed to explain the roles of the a,6, and E DNA polymerases in eukaryotic DNA replication (lo),and more may have to be devised. It has become dogma that DNA polymerase a synthesizes only short RNA-DNA primers, thus making only a small quantitative contribution to total DNA synthesis. Nucleotides misincorporated by DNA polymerase a might be corrected by the 6 and E 3 ' 4 5 ' exonucleases, or might simply result in abortive priming. From the spontaneous mutation rates of the PO&01 and pd2-4 mutants, the 3 ' 4 3 ' exonuclease of the 6 polymerase appears to make an approximately 10-fold greater contribution to reducing spontaneous mutations than does that of the E polymerase. Whether this reflects the relative contributions of the two polymerases to DNA synthesis depends on their ratios of exonuclease and polymerase activities in the cell, which are unknown. The locations of the spontaneous mutations generated in the pol% 4 and POD-01 mutants suggest that both polymerases act at sites scattered widely throughout the genome, and both act within the URA3 gene inserted near the ARS306 replication origin. These results are consistent with models in which both E and 6 DNA polymerases participate in DNA replication, but further investigation is needed to define their precise roles.

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GLOSSARYOF YEAST GENESO ADE2 ADE5 ARS306 CAN1 CDC2 CDC4 CDCl7

DPB2 DPB3

nm HIS7

HO HOM3 HSP6O LEU2

LYSI

LY s2 MAT MlPl PMSl

POL1

POL2 POL2 POLX

Encodes phosphoribosylaminoimidazole carboxylase; its mutants are &nine auxotrophs Encodes phosphoribosylglycinamide synthetase; its mutants are adenine auxotrophs Locus of DNA replication origin 306 on chromosome 111 Encodes arginine permease; confers sensitivity to canavanine Required for progression of the _cell-_division_cycle, encodes DNA polymerase 111 (Table I) and is identical to POL3 Required for progression of the cell-division zycle Required for progression of the cell-division _cycle; encodes DNA polymerase I (Table I) and is identical to POL1 Encodes subunit B of DNA polymerase I1 (Table 11); inutants are listed in Table 111 Encodes subunit C of DNA polymerase I1 (Table 11); mutants are listed in Table 111 Encodes ATP phosphoribosyltransferase; mutants are histidine auxotrophs Encodes glutamine amidotransferase; mutants are &tidine auxotrophs Directs efficient interconversion of mating type in homothallic strains Encodes aspartate kinase; mutants are k o s e r i n e auxotrophs Encodes a mitochondrial heat-qhock protein Encodes P-isopropylmalate dehydrogenase; its mutants are leucine auxotrophs Encodes saccharopine dehydrogenase; its mutants are k i n e auxotrophs Encodes 2-aminoadipate reductase; its mutants are k i n e auxotrophs Locus determining the E t i n g type of a haploid cell; present as either a or 01 Encodes mitochondrial DNA polymerase (see Table I) Required for mismatch repair; gene product shows aminoacid sequence similarity to the prokaryotic mutL and h e d mismatch-repair genes; p r m l strains display a spontaneous mutator phenotype in mitotic cells and, in meiosis, show increased post-meiotic segregation of mutations Encodes catalytic subunit of DNA wlymerase I (Table I); identical to C D C I 7 Encodes catalytic subunit A of DNA p&ymerase 11 (Tables I and 11); mutants are listed in Table 111 Encodes catalytic subunit of DNA polymerase 111 (Table I); identical to CDC2 Encodes a “computer DNA polymerase” with amino-acid sequence similarity to higher eukaryotic DNA polymerase p (Table 1)

117

YEAST DNA POLYMERASE GLOSSARY(continued) RAD6

Required for the repair of radiation-damaged DNA; RAD6 is the eponymous member of a group of genes that includes REV3 and RAD18, and its mutants have a pleiotropic phenotype; encodes a ubiquitin conjugase and is identical to

RAD18 RA DS0 REV3

Required for the repair of +iation-damaged D N A Required for the repair of radiation-damaged D N A Required for the appearance of genomic D N A mutations following treatment of cells with genotoxic agents, a phenotype typically measured by reversion of auxotrophic marker genes; encodes a “computer DNA polymerase” with amino-acid sequence similarity to class-B D N A polymerases (see Table I) Encodes uracil D N A glycosylase Encodes orotidine-:,‘-phosphate decarl~oxylase;its mutants confer uracil auxotrophy and resistance to 5-fluoroorotic acid

UBCZ

UNGl uRA3

“By convention, S. cerecisiae gene symbols consist of three letters followed by a number, all italicized. Upper-case letters, as in “POLi?,” signify dominance, while lower-case letters are used for recessive genes. Mutants ofa gene are given allele nnmberc, as in “,no&18,” nr the symbol “A,” which indicates “deletion,”or the letters “fs,” indicating “temperature-sensitive.”Information on the genes listed is from 121-123 or from references quoted in the text.

ACKNOWLEDGMENTS The authors are grateful to D. Thomas, P. Ropp, and C. Bennet for their many comments on this manuscript, and to J. E. Syvaoja and Z. Wang for communicating unpublished data.

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