Mol Gen Genet (1994) 242:289-296

© Springer-Verlag 1994

The 5' exonucleases of both D N A polymerases J and e participate in correcting errors of D N A replication in Saceharomyces cerevisiae Alan Morrison ~, Akio Sugino 2

1 Laboratory of Molecular Genetics° National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC27709, USA 2 Department of Molecular Immunology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565, Japan

Received: 10 June 1993/Accepted: 21 July 1993

Abstract. DNA polymerases II (e) and III (fi) are the only nuclear DNA polymerases known to possess an intrinsic 3'-~5' exonuclease in Saccharomyces cerevisiae. We have investigated the spontaneous mutator phenotypes of DNA polymerase J and e 3'---5' exonuclease-deficient mutants, pol3-O1 and po12-4, respectively, pol3-O1 and po12-4 increased spontaneous mutation rates by factors of the order of 102 and 101 , respectively, measured as URA3 forward mutation and hisT-2 reversion. Surpris- ingly, a double mutant po12-4 po13-O1 haploid was invi- able. This was probably due to accumulation of unedited errors, since a po12-4/po12-4 po13-O1/po13-01 diploid was viable, with the spontaneous hisT-2 reversion rate in- creased by about 2 x 103-fold. Analysis of mutation rates of double mutants indicated that the 3 ' ~ 5 ' exonucleases of DNA polymerases fi and e can act competitively and that, like the 3'-~5' exonuclease of DNA polymerase J, the 3 ' ~ 5 ' exonuclease of DNA polymerase e acts in se- ries with the PMS1 mismatch correction system. Muta- tional spectra at a URA3 gene placed in both orienta- tions near to a defined replication origin provided evi- dence that the Y ~ 5 ' exonucleases of DNA polymerases J and e act on opposite DNA strands, but were in suffi- cient to distinguish conclusively between different models of DNA replication.

Key words: DNA polymerases e and J 3 ' ~ 5 ' Exonucle- ase - Replication errors - Spontaneous mutations - Sac- charomyces cerevisiae

Introduction

Prokaryotic DNA polymerases possess a tightly asso- ciated 3'-~5' exonuclease whose role is to edit out incor- rectly inserted nucleotides and thus enhance accuracy of

Communicated by M. Sekiguchi Correspondence to: A. Sugino

DNA replication (Echols and Goodman 1991). In eu- karyotes, as exemplified by the yeast Saccharomyces cerevisiae, five nuclear DNA polymerases have been ob- served or predicted: DNA polymerases I(~), II(6), III(~), IV(J3) and the putative REV3 DNA polymerases (Wang 1991 ; Morrison and Sugino 1993). Current evidence sug- gests that only DNA polymerases a, g and ~ participate in DNA replication (Morrison and Sugino 1993). Puri- fied DNA polymerases e and g both possess a tightly associated 3'--.5' exonuclease proofreading activity, while five active site residues of an evolutionarily con- served 3 ' - ,5 ' exonuclease domain have been identified in the N-terminal regions of their catalytic subunits (Mor- rison et al. 1991; Simon et al. 1991). The structurally related ~ DNA polymerase and the putative REV3 DNA polymerase, however, lack these 3'-~5' exonuclease ac- tive site residues (Morrison et al. 1991; Morrison and Sugino 1993). The human DNA polymerase ~ catalytic subunit is devoid of 3 ' ~ 5 ' exonuclease activity (Copeland and Wang 1991), as is the yeast DNA poly- merase a holoenzyme (Kunkel et al. 1989). DNA poly- merase 13 of higher eukaryotes lacks both the conserved 3 ' ~ 5 ' exonuclease domain and an intrinsic 3'--,5' exonu- clease activity (Matsukage et al. 1987; Linn 1991); al- though a nuclease, exonuclease V, is sometimes asso- ciated with DNA polymerase 13, it does not have the properties of an editing exonuclease (Linn 1991). DNA polymerases ~ and a therefore appear to be the only known nuclear DNA polymerases capable of performing 3'--*5' exonucleolytic editing.

Mutation of conserved 3 ' ~ 5 ' exonuclease active site residues of the S. cerevisiae DNA polymerases ~ or ~ reduces the ratio of exonuclease:polymerase activities of the partially purified enzymes by at least 100-fold, and causes a spontaneous mutator phenotype with no conco- mitant cell growth defect (Simon et al. 1991; Morrison et al. 1993, 1991). The mutation rate increase is about 130- to 400-fold in the case ofpol3 (Simon et al. 1991; Morrison et al. 1993). The DNA polymerase ~ 3 ' - ,5 ' exonuclease corrects a broad spectrum of single-base mutations and acts in series with the PMS1 mismatch

290

correction system so that their effects are multiplied (Morrison et al. 1993). Such data provide evidence for a proofreading role of the 3 '~5 ' exonuclease of DNA polymerase ~ in vivo.

Based on an examination of epistatic relationships, we propose here that the DNA polymerase e 3 '~5 ' exonu- clease also participates in proofreading in vivo. The total contribution of both Y ~ 5 ' exonucleases of DNA poly- merases 6 and ~ to the reduction of spontaneous muta- tions appears to be of the order of 103-fold, and a haploid deficient in both activities is inviable. We also compare the mutational spectra of the 3' ~ 5' exonuclease-deficient DNA polymerases ~ and e mutants, and discuss the results in terms of the roles of the DNA polymerases in DNA replication.

Materials and methods

Yeast strains. Yeast strains were: CG379 (MA Tc~ ade5-1 his7-2 leu2-3,-112 trpl-289 ura3-52), CG378 (MATa ade5-1 canl leu2-3,-112 trpl-289 ura3-52), AMY360-8D (MATa pol3-O1 his7-2 ade5-1 leu2-3,-112 trpl-289 ura3- 52) (Morrison et al. 1993) and AMY360-11A (MATa Ieu2-3,-112 trp l-289 ura3-52 ade5-1 his7-2 pol3-O1).

Plasmids. pBL304 (11.05kb), containing the 3.7kb MluI-HindIII interval of POL3 DNA cloned as a SAll- HindIII fragment into the SaII-HindIII sites of YCp50, was constructed by P.J.M. Burgers. pBLAM1 was de- rived from pBL304 by replacement of POL3 by pol3-O1 (Morrison et al. 1993). PAM140 was derived from pBLAM1 by replacement of a 1.5 kb NruI-SmaI interval containing URA3 with a 2 kb HpaI fragment from YEp13 containing LEU2 in the orientation opposite to that of pol3-O1. PAM150, a pol2-4 TRP1 CENIV plas- mid, was derived from YCpPOL2 (Araki et al. 1992) by replacement of the POL2 MluI-BamHI fragment with the corresponding poi2-4 fragment from YIpJB1 (Mor- rison et al. 1991).

Construction of yeast strains. Construction of the pol2-4 andpoI3-O1 derivatives of CG379 and insertion of URA3 (in the LR orientation, i.e. the URA3 coding sequence is the lagging strand relative to the ARS306 replication origin) about 4.4 kb distal to the ARS306 replication origin (Reynolds et al. 1989) were described previously (Morrison et al. 1991, 1993). Insertion of URA3 in the opposite RL orientation followed the same procedure; transplacement of the 1.1 kb URA3 BamHI fragment into the BglII interval ofARS306 DNA ofPAM81 (Mor- rison et al. 1993) naturally generated both orientations. Activity of the ARS306 origin was confirmed by 2-D gel electrophoresis of replicating DNA (Brewer and Fang- man 1987) from several different strains (unpublished observations). A pmsl mutation was introduced into CG379 and its pol2-4 derivative by transplacement of the coding region of the chromosomal PMS1 gene by LEU2 using plasmid PAM58 (Morrison et al. 1993). Diploids used in Table 2 were derived from crosses of CG379 and CG378 or their pol2-4, pol3-O1 or pmsl derivatives.

PMS1 segregants from a cross of CG379 and CG378 pmsl were crossed with CG379 to make the control + / + + / + diploid. AMY360-SD, a pol3-O1 segregant obtained from a cross of CG379 pol3-O1 and CG378, was crossed with CG379 pol2-4 to make diploid AMY410. AMY410 was transformed with the POL3 URA3 plas- mid pBL304, sporulated, and segregants were crossed t(; make the other diploids listed in Table 2. These diploids were plated on 5-fluoroorotic acid (FOA) to remove pBL304 before the his7-2 reversion measurements (Boeke et al. 1984).

Yeast methods. Measurement of spontaneous mutation rates and genotyping of pol3-O1 using the polymerase chain reaction (PCR) were as described (Morrison et al. 1993). To determine poi2-4 genotypes, a 0.28 kb segment of POL2 DNA was amplified from yeast genomic DNA by PCR with 30 cycles of 94 ° C, 42 ° C, 72 ° C using an oligonucleotide primer matching the complement of bases 1705-1730 (Morrison et al. 1990) and either oligonucleotide 5'-AATGGCATTTGATATAGA-3', matching nucleotides 1449-1466 of POL2 + (Morrison et al. 1990), or 5'-AATGGCATTTGCTATAGC-3', matching the equivalent pol2-4 sequence. The latter two primers each generated the expected 0.28 kb DNA frag- ment only with their cognate template DNA. A PCR test, using 30 cycles of 94 ° C, 52 ° C, 72 ° C, was employed to check the orientation of URA3 integrated near to ARS306. Reactions contained an oligonucleotide primer corresponding to nucleotides 69539-69560 of chro- mosome III (Oliver et al. 1992) and either primer P4 representing nucleotides 785-805 of URA3 (Rose et al. 1984) or primer P8 representing the complement of URA3 nucleotides 475-455. With URA3 in the LR orientation (see Fig. 1 of Morrison et al. 1993), primer P4 generated an approximately 430 bp PCR DNA frag- ment; with URA3 in the RL orientation, primer P8 generated an approximately 520 bp PCR DNA frag- ment,

Nucleotide sequence of ura3 mutations. The URA3 gene of 5-fluoroorotic acid-resistant (FOA R) mutant yeast cells was amplified by PCR and sequenced as described (Morrison et al. 1993). For URA3 in the LR orientation, 20/31 pol3-O1 and 20/33 po12-4 ura3 mutations were de- termined for strains cultured in YPDA medium. Similar- ly, 20/36 pol3-O1 and 13/23 pol2-4 ura3 mutations with URA3 in the RL orientation were determined for strains cultured in YPDA medium. The remainder were cultured in synthetic complete medium, since it was found that the spontaneous FOA R mutation rate of the control strain was lower in this medium (our unpublished results). From the relative (to the control strain) ura3 mutation rate of about 130 for pol3-O1 in YPDA medium, we expect that none of the sequenced pol3-O1 mutations in Fig. 2 is a background mutation (Lee et al. 1988). Since the relative mutation rate forpol2-4 is about 12 in YPDA medium and about 86 in synthetic complete medium, it is likely that about 2/33 sequenced mutations are due to background for URA3 in the LR orientation, and about 1/23 in the RL orientation. For the first 20 pol3-O1 and

20 pol2-4 mutations, the coding region of the ura3 gene was sequenced completely, as described (Morrison et al. 1993); this showed that only one, single-base mutation occurred in each case. For the remaining mutations, sequencing was continued only until a mutation was found. After sequencing, the POL2 and POL3 genotypes and URA3 orientations of the source DNAs were checked in selected cases to rule out errors.

Resu l t s

Spontaneous mutation rates in po13-01 and po12-4 strains

We described previously the construction of exonuclease- deficient pol2-4 and po13-01 mutants by mutating both Asp and Glu residues to Ala residues in the exonuclease I domain, which is well conserved among DNA poly- merases possessing the Y ~ 5 ' exonuclease activity

EXQ I

A A $ 1"

S. cerevisiae Pole 286-V M A F ~:i .... I ~ T T K-295

S. cerevisiae Pol~ 31V-I M S F ~i I ~i C A G - 3 2 6 iiiiiiii iiiiiiii

S. pombe Pole 265-V M A F ~i I ~ T T K-274 ........ iii=:=:iii

Human Pole 244-V L A F ~i I ~ T T K-253

Human Po16 312-V L S F ~i I ~ C A G-321

Herpes simplex 364-L M C F ~i I ~ C K A-373

E. coli PolII 152-W V S I ~] I ~ T T R-161

T4 108-V A N C ~ I ~ V T G-II7

¢029 8-M Y s c ~ ~" ~ T T T-17

Fig. 1. Alignment of DNA polymerase amino acid sequences proposed to contain conserved 3'-+5' exonuclease active site resi- dues. The one-letter code is used. Numbers refer to amino acid residues. One of the three conserved regions (Exo I) is shown. Shaded amino acids indicate the residues proposed to correspond to the 3 ' ~ 5 ' exonuclease active site residues D355 and E357 of Escherichia col± polymerase I Klenow fragment. Arrows indicate the mutational changes of D N A polymerases 6 and e employed in this study. Sequences are from the GenBank/EMBL database. The Schizosaccharornyces pornbe DNA polymerase a sequence is the unpublished work of J. Sebastian and A. Sugino. Saccharomyces cerevisiae D N A polymerase 6 is numbered according to Morrison and Sugino (1992a)

291

(Fig. 1) and insertion of the URA3 gene (in the LR orientation) near to the ARS306 replication origin on S. cerevisiae chromosome III (Morrison et al. 1993). Spon- taneous URA3 and his7-2 mutation rates are given in Table 1. The relative (to wild type) URA3 mutation rate forpol3-01 in strain CG379 was 130, and the correspond- ing value for pol2-4 was 12 (Table 1). The latter value is in the same range as 20, the average of the relative reversion rates determined for six different markers: his7- 2 (Table 1), ade2-1 (unpublished observations), and ade5- 1, his1-7, leu2-1 and horn3-10 (Morrison et al. 1991).

Multiplicative relationship between pmsl and po12-4

A pol2-4 pmsl strain was viable but showed poor growth and heterogeneous colony size. It had relative mutation rates of about 520 and 1800 at URA3 and his7-2, respec- tively (Table 1), reasonably close to 490 (12x 41) and 1400 (9.3 x 150), the respective minimum values expected of a multiplicative relationship between these two mu- tants. Multiplicity is expected if the DNA polymerase 3 '~5 ' exonuclease and PMS1 act in series on the same potential mutations.

Different colonies from one of two isolates of the pol2-4 pmsl strain initially tested gave quite different URA3 mutation rates: either values similar to those in Table 1 or considerably lower - about the same as for pros1 (data not shown). On reconstruction of the strain, all three independent isolates tested displayed the high mutation rates given in Table 1. It therefore appears that suppressors of the extremely high mutation rate can appear, but we have not investigated this further.

The po12-4 po13-01 haploid is inviable

We attempted unsuccessfully to introduce the pol3-O1 mutation into the CG379 pol2-4 strain (data not shown), leading us to suspect that the pol2-4 and po13-01 muta- tions are incompatible. We tested this by tetrad analysis of spores from diploids heterozygous for both po13-01 andpol2-4. Thirty-six tetrads from the diploid AMY360- 8D x CG379 po12-4 yielded 4:0, 3 : 1, and 2:2 segregation of viable :inviable spores in the ratio 1.1:3.9:0.9, close to the ratio 1:4:1 expected theoretically for incompatibility

Table 1. Spontaneous mutat ion rates in haploids

Genotype Forward mutat ion of Ura3"

Mutat ion rate ( x 108) b Relative rate

Reversion of his7-2

Mutat ion rate (× 108) Relative rate

Wild type 1.8± 0.67 (4) ° 1 po12-4 22 _+ 9.3 (5) 12 pmsl 74 _+ 36 (3) ° 41 pol3-O1 240 ± 97 (3) ~ 130 pol2-4pmsl 940 ±410 (5) 520

0.55-t- 0.23 (15) ° 1 5.1 ± 2.3 (9) 9.3

85 ± 27 (12)o 150 130 _ 30 (9) c 240

1000 +530 (7) 1800

a URA3 gene inserted near the ARS306 D N A replication origin in the LR orientation b Isogenic derivatives of CG379 were cultured in YPDA medium.

Results given as mean _+ standard deviation (number of determina- tions) c Data from Morrison et al. (1983)

292

÷ - - 0 0 0 + - 0 0 + ÷ - 0 - - 0 0 - ÷ - ÷ 0 ÷ ÷ - ÷ ÷ - ÷ ÷ ÷ 0 ÷ ÷ ÷ - + ÷ - ÷ ÷ - ÷ - - 0 0 0 - ÷ ÷ - ÷ ÷ - + + + - - ÷ ÷ + ÷ 0 ÷ + ÷ 0 + -

POL3

- ÷ + 0 0 0 - ÷ 0 O - ÷ ÷ O ÷ + O 0 ÷ - ÷ ÷ 0 - - ÷ ÷ ÷ ÷ ÷ ÷ ÷ 0 - ÷ ÷ ÷ - - + ÷ + ÷ - ÷ + 0 0 0 + + + + - - + - - ÷ + ÷ - - - + 0 - - - 0 - +

POL2

Fig. 2. Tetrad analysis of diploid heterozygous for pol2-4 and pol3-O1. A photograph is shown of spores microdissected from tetrads of AMY360-11A (pol3-O1)x CG379 pol2-4 and grown on YPDA plates. Genotypes are diagrammed below: +, wild type; - , mutant (pol2-4 or pol3-O1); o, no visible colony

Table 3. Epistatic relationship between pol2-4 and pol3-O1

Genotype Mutation rate Relative rate (X lOS) a

Wild type 0.28 4- 0.21 (3) 1 pol2-4 24 + 7.6 (3) 86 POL3+[pol3-O1] b 16 i3 .8 (3) 57 pol2-4POL3+[pol3-O1] b 51 ±4.8 (3) 180 POL2+[pol2-4] ° 4.6 ± 1.0 (2) I6 pol2-4 [POL2+] d 4.4 ± 1.5 (2) 16

" Spontaneous mutation was measured as forward mutation of the URA3 gene inserted near the ARS306 DNA replication origin in the LR orientation. Isogenic derivatives of CG379 were grown in syn- thetic medium lacking either tryptophan or leucine to select for plasmid maintenance. Results given as mean ± standard deviation (number of determinations) b The pol3-O1 plasmid was PAM140 c The pol2-4 plasmid was PAM150 d The POL2 plasmid was YCpPOL2 (Araki et al. 1992)

Table 2. Spontaneous mutation measured as reversion of his7-2 to His + in diploids

Genotype Reversion of his7-2

Mutation rate (x 108) ~ Relative rate

+ / + + / + 0.37_+ 0.16 (6) b,c ] +/+ po12-4/po12-4 3.7 _+ 0.2 (2) a 10 pol3-O1/pol3-O1 + / + 180 _+ 120 (4) e,° 490 pol3-O1/pol3-O1 690 -t- 50 (3) f 1900 po12-4 /po12-4

a Results given as mean +_ standard deviation (number of determina- tions), or mean_+ range when only two determinations were made b Two determinations for each of three different strains c Data from Morrison et al. (1993) d Deteminations for two strains e Two determinations for each of two strains f Determinations for three strains

(data no t shown). Similar data were obta ined using dip- loid A M Y 3 6 0 - 1 1 A x C G 3 7 9 pol2-4 crosses (Fig. 2; unpubl ished observations). Genotyp ic analysis o f these spores showed no viable spore with the inferred pol2-4 po13-01 genotype (Fig. 2). In controls, bo th pol2-4 and po13-01 segregated 2:2 (not shown). The pol2-4 po13-01 spores germinated and formed microcolonies o f abou t 50 cells (average o f 18 determinations). These cells had no unique terminal morpho logy , and consisted of, on average, approximate ly 50% single, unbudded cells, 8% budded cells, 28% dumb-bells, 4% aberrants, and 10% others.

po12-4/po12-4 po13-01/po13-01 diploid

There are two plausible explanations for the inviability o f the pol2-4 pol3-O1 haploid. First, a D N A polymerase

Y-+ 5' exonuclease is required for cell cycle progress ion; this is no t likely, given the absence o f a unique terminal morpho logy , and would no t be corrected by diploidy. Second, the unedited replication errors are lethal to the haploid cell; in this case, diploidy might restore viability.

We constructed a po12-4/po12-4 pol3-O1/pol3-O1 dip- loid by mat ing haploids o f genotype po12-4 pol3-O1 [POL3 URA3] (i.e. the POL3 and URA3 genes were present on plasmid pBL304). Three such diploids were constructed f rom different haploid segregants o f AMY410. The diploids were g rown in non-selective YP- D A medium and plated on F O A to select for loss o f the POL3 URA3 plasmid. The median plating efficiency on F O A of ten independent cultures o f each o f the three diploids was 0.06 4- 0.03. We confirmed the po12-4/po12-4 pol3-1/pol3-O1 genotype o f five F O A R isolates o f the diploids using P C R tests, and concluded that the double homoallelic diploid is viable and thus that a D N A poly- merase 3 ' -+5 ' exonuclease is not required for cell cycle progression. We measured the relative his7-2 reversion rate o f the double homoa lMic diploids as 1 .9x 103 (Table 2).

Fig. 3. Mutational spectra in the URA3 gene in both LR and RL orientations in the po13-O1 and pol2-4 derivatives of CG379. The URA3 nucleotide sequence is shown, numbered from the first nu- cleotide of the start codon. Above the sequence are mutations with URA3 in the LR orientation (i.e. the URA3 coding sequence is the lagging strand relative to the ARS306 replication origin). Below the sequence are mutations with URA3 in the RL orientation (i.e. the URA3 coding sequence is the leading strand), pol3-O1 base sub- stitutions are in bold, upper-case letters, pol2-4 base substitutions are in lower-case letters. Single-base deletions are open lozenges and trianoles for pol3-O1 and pol2-4, respectively. Single-base insertions are closed lozenges and triangles for pol3-O1 and pol2-4, respectively; the base inserted is the same as that marked by the symbol. When the deletion or insertion occurred within a homooligomeric run, the symbol is arbitrarily positioned at the first nucleotide of the run

¢.

A T G TCG A A A GCT A C A T A T AAG G A A CGT GCT GCT A C T C A T CCT AGT C C T G T T GCT GCC AAG C T A T T T A A T A T C ATG 75

C C C C

O C C O C CAC GAA AAG C A A A C A A A C T T G T G T GCT T C A T T G G A T G T T CGT ACC ACC AAG GAA T T A CTG GAG T T A G T T GAA GCA

• . C a t ¢ T O t A

a a a G

V A G • a V A C G C t • t

T T A GGT CCC A A A A T T T G T T T A C T A A A A A C A C A T GTG G A T A T C T T G A C T GAT T T T TCC ATG GAG GGC A C A G T T AAG O A CTg CA

g a

150

225

G t aC c

CCG C T A AAG GCA T T A TCC GCC AAG T A C A A T T T T T T A CTC T T C GAA GAC AGA A A A T T T GCT GAC A T T GGT A A T A C A c T O C T

O

300

a t

a T t GTC A A A T T G CAG T A C T C T GCG GGT G T A T A C AGA A T A GCA GAA TGG GCA GAC A T T ACG A A T GCA CAC GGT GTG GTG

T a T T

375

a a

GGC CCA GGT A T T G T T AGC GGT T T G AAG CAG GCG GCA GAA GAA G T A A C A AAG GAA CCT AGA GGC C T T T T G A T G T T A a T a

a a a g

GCA GAA T T G T C A TGC AAG GGC TCC C T A T C T A C T GGA GAA T A T A C T AAG GGT A C T G T T GAC A T T GCG AAG AGC GAC a

450

525

A t • A A A G A T T T T G T T A T C GGC T T T A T T GCT C A A AGA GAC A T G GGT GGA AGA GAT GAA GGT T A C GAT TGG T T G A T T A T G

C. 600

a ¢ A C A CCC GGT GTG GGT T T A G A T GAC A A G GGA GAC GCA T T G GGT C A A CAG T A T AGA ACC GTG G A T G A T GTG GTC T C T 675

a a ¢ T ¢ T

t t t t

t t t t t t A

¢ t A a •

A C A GGA T C T GAC A T T A T T A T T G T T GGA AGA GGA C T A T T T GCA AAG GGA AGG G A T GCT AAG G T A GAG GGT GAA CGT 750 T t a 0 T ¢ T 0

A ¢ a • ¢

T A C AGA A A A GCA GGC TGG GAA GCA T A T T T G AGA AGA TGC GGC CAG C A A A A C T A A a t A A g

804

294

Table 4. Classes of spontaneous mutation in the URA3 gene in both LR and orientations in the po13-Ol and pol2-4 derivatives of CG379

Class Mutation pol3-O1 pol2-4 URA3 (LR) URA3 (RL) Combined URA3 (LR) URA3 (RL) Combined

Transitions

Transversion

Deletions

Insertions

Total

G.C~A.T 4 4 8 7 2 9 A.T-,G.C 9 2 11 0 1 1

G.C--+C.G 0 0 0 0 0 0 G.C~T.A 1 9 10 3 5 8 A.T ~T.A 2 4 6 19 8 27 A.T~C.G 4 2 6 1 4 5

- 1 7 13 20 2 0 2

+1 4 2 6 1 3 4

31 36 67 33 23 56

Epistatic relationship between po13-01 and po12-4

A synergistic relationship, i.e. the effect of the double mutation is greater than the sum of the single mutations, between pol3-O1 and pol2-04 is apparent for both the his7-2 mutation rate (Table 2) and growth (the single mutations caused no growth defect, while the double mutation caused inviability). There are two possible rela- tionships that result in synergy: action in series, and competition for the same pool of replication errors. (These relationships are illustrated in Fig. 3 of Morrison et al. 1993, together with the predicted spontaneous mutation rate of the double mutant, Mdp, as a function of those of the individual mutants, Ma and Mp.) Simply measuring the spontaneous mutation rate of the double mutant will not necessarily distinguish between these two relationships. They can be distinguished, however, by inclusion of one of the wild-type DNA polymerase genes, here POL3, in thepol2-4pol3-O1 mutant. This will reduce synergy in the case of competition to simple additivity, but will preserve multiplicity in the case of action in series, pol3-O1 is partially dominant, so that the POL3/ pol3-O1 combination has a measurable mutator effect (Morrison et al. 1993). (Similarly, pol2-4 is also partially dominant, whether the pol2-4 allele is chromosomal or on a CEN plasmid; Table 3.) Therefore, we compared the relative mutation rates of pol2-4, POL3 pol3-O1, and pol2-4 POL3 pol3-O1 strains. The pol3-O1 gene was on plasmid PAM140 introduced into isogenic POL3 and pol2-4 strains, and forward mutation of URA3 was mea- sured (Table 3). The observed relationship was close to additive: the calculated additivity of the relative muta- tion rates of POL3 pol3-O1 and pol2-4 strains was 5 7 + 8 6 - 1 = 142, within experimental error of the 180 measured for the pol2-4 POL3 pol3-O1 strain, and 35-fold lower than 57 x 86 = 4900, the minimum value expected of a mutiplicative relationship (Morrison et al. 1993). This contrasts with the result of a conceptually similar experiment with the pol3-O1 and pmsl mutants, where multiplicity was preserved (Morrison et al. 1993).

The values in Table 3 were for strains cultured in synthetic medium. The spontaneous FOA R mutation rate for the pol2-4 strain grown in synthetic medium, 2.4x 10 -7, was about the same as the 2.2x 10 - 7 ob-

tained when growth was in YPDA medium (Table 1). However, the value for the control strain was apparently about sixfold lower, and therefore the relative rate for the pol2-4 strain was correspondingly higher.

Nucleotide sequence of FOA R mutations

We sequenced limited numbers of independent ura3 mutations in the pol3-O1 and pol2-4 strains with URA3 inserted close to ARS306 in both (LR and RL) orienta- tions. Reversing the orientation of URA3 did not ap- preciably change the spontaneous URA3 mutation rates of po12-4 and pol3-O1 strains: with URA3 in the RL orientation, the pol2-4 and pol3-O1 FOA R mutation rates were l a x 10 -7 and 2x 10 -6, compared to 2.2x 10 -7 and 2.4x 10 -6, respectively, with URA3 in the LR orientation (Table 1). The mutations are classified in Table 4. All classes of single-base mutation were ob- served in pol3-O1 and pol2-4 strains, except for G . C ~ C . G transversions. In the data set, single-base deletions were more frequent in pol3-O1 than in po12-4, and base substitutions were heavily biased in favor of transversions in pol2-4 but not in pol3-O1. The mutation- al spectra are shown in Fig. 3, and are discussed below.

D i s c u s s i o n

Synergy between po13-01 and po12-4

Synergy was observed between the pol3-O1 and po12-04 mutations both for growth and for his7-2 reversion (Table 2). The synergy of mutation rates was shown to reflect a competitive relationship (Table 3), indicating that both exonucleases can act to correct the same pool of potential mutations. Presumably, nucleotides misin- corporated by a polymerase are corrected preferentially by its intrinsic 3'---,5' exonuclease, so that the com- petition is normally only potential. The results suggest the possibility that the DNA polymerase 6 and/or exonucleases may remove nucleotides misincorporated by DNA polymerase 4, which has no intrinsic proofread- ing 3 ' ~ 5 ' exonuclease. Interestingly, a search for an

295

autonomous 3 '~5 ' proofreading exonuclease activity that could edit errors committed by DNA polymerase found an activity identified as DNA polymerase 6 (Per- rino and Loeb 1990).

Contribution of exonucleolytic editing to DNA replication fidelity

Synergy between pol3-O1 and pol2-4 suggests that the contribution of exonucleolytic proofreading to DNA replication fidelity is greater than expected from the rela- tive mutation rates of the respective single mutants. A rough approximation of the minimum contribution is given by the 1.9 x 103 value for the his7-2 relative muta- tion rate in a pol3-O1/pol3-O1 po12-4/po12-4 diploid (Ta- ble 2). Inviability of the po13-01 po12-4 haploid indicates that there must be at least one event per cell division. An increase of 2 x 103 in the spontaneous genomic mutation rate translates to about six genomic mutations per cell division (Drake 1991). These mutations are cumulative with each cell division, and should be lethal after several divisions.

It may be objected that the excessively high mutation rate of the po13-01 pol2-4 strain reflects saturation of the mismatch repair system in the absence of exonucleolytic editing, as occurs with the dnaQ mutation in Escherichia coli (Schaaper 1988). The hallmark of such saturation is that the product of the relative mutation rates of the exonuclease-deficient and mismatch repair single mu- tants (i.e. Md x Mp) is considerably greater than the value (Mdv) measured for the double mutant. Conversely, in the absence of saturation, the prediction is that Mdp_>MdXMp (Morrison et al. 1993); this is the ob- served result for his7-2 reversion and URA3 mutation with po13-01 and pmsl (mismatch repair) mutants in S. cerevisiae (Morrison et al. 1993).

Model for DNA replication error correction

We have discussed a model for the DNA replication/ error correction cycle in which DNA is replicated by DNA polymerases a, 5 and s, misincorporated nucleo- tides are corrected by the 3'-~5' exonucleases of DNA polymerases g and s, and remaining errors are corrected by the serial action of the PMS1 mismatch correction system (Morrison and Sugino 1992b). The epistatic rela- tionships between the po13-01, pol2-4 and prnsl genes shown here and previously (Morrison et al. 1993) form the framework for this error correction scheme. Thus, PMS1 acts in series with both the 3'-~5' exonucleases of DNA polymerases 6 and s which themselves are in a (potentially) competitive relationship.

Roles of DNA polymerases in DNA replication

Current models presume that DNA polymerase ~ makes short RNA-DNA primers at replication origins and on the lagging strand, and that the rest of the DNA is

replicated by DNA polymerases 8 and ~. In one model, DNA polymerase 8 replicates most of both DNA strands, while DNA polymerase ~ fills in any remaining gaps on the lagging strand (Kunkel 1992). In another model, one DNA polymerase replicates the leading strand and the other the lagging strand (Morrison et al. 1990). The observation that the spontaneous mutator phenotype ofpol2-4 is only about 10% as strong as that ofpol3-01 (Table 1) is apparently more consistent with the first model, but this is inconclusive because the ratio of 3 '~5 ' exonuclease to polymerase activity for DNA polymerases ~ and s is not known. Furthermore, it has been suggested that at least two DNA polymerases, DNA polymerase a and either DNA polymerase 6 or s, participate in the formation of RNA-linked Okazaki fragments during DNA replication (Morrison et al. 1990; Nethanel and Kaufman 1990). Therefore, it is possible that the amount of DNA synthesized by DNA polymerase a may be very small and DNA synthesized by the polymerase may be further removed by the nu- clease when RNA primers are degraded. If this were the case the cell can achieve a high fidelity of DNA synthesis without editing by DNA polymerase a as long as two other DNA polymerases have the 3 '~5 ' exonuclease activity.

We suggest that the spontaneous mutations generated in the pol3-O1 and pol2-4 strains may be taken as qualita- tive indicators of the sites of action of DNA polymerases

and e, respectively. By this measure, both DNA poly- merases act at sites widely scattered throughout the genome. More interesting are the mutational spectra at the URA3 gene placed in two orientations close to the ARS306 origin. The rationale is as follows: because mutational spectra depend on the nucleotide sequence of the template DNA, an exonuclease-deficient DNA poly- merase replicating the URA3 coding strand should generate a quite different spectrum from that observed when it replicates the non-coding strand. If DNA poly- merase 6 replicates essentially all of both strands, the observed mutational spectrum will be the sum of muta- tions on both coding and non-coding strands; when the orientation of URA3 is reversed, the po13-01 mutational spectrum should remain essentially the same. Of course, some differences might be expected because of mutations generated by, say, DNA polymerase ~. Conversely, if DNA polymerases 5 and ~ replicate, say, the leading and lagging strands, respectively, then reversing the orienta- tion of URA3 should completely change the po13-01 and pol2-4 spectra, but the spectrum generated by one DNA polymerase with URA3 in one orientation should resem- ble the spectrum generated by the other DNA poly- merase with URA3 in the opposite orientation. The latter spectra, however, would not be expected to be identical because of the different identities of the polymerases.

In Fig. 3, which shows the po13-01 and pol2-4 muta- tional spectra at URA3 in both orientations relative to the ARS306 origin, all four spectra appear to be different, but there are nine sites at which the same mutation occurs in more than one spectrum. (We include in the definition of site, homooligomeric runs where a single- base frameshift occurs.) Of these nine cases, one

296

(A686~T) represents pol2 mutations occurring in both URA3 orientations, one (T 166 ~ C) represents pol3 muta- tions occurring in both URA3 orientations, two (T164~A and G764-,A) represent a pol3 mutation oc- curring in one URA3 orientation and both pol3 and pol2 mutations in the other orientation, and five (A160A, T168---,G, +T183, A279-,T and G345~T) represent poI3 mutations occurring in one URA3 orientation and pol2 in the other. These results appear to provide provi- sional evidence that DNA polymerases 5 and e can act on opposite DNA strands. This, however, does not necessarily distinguish between the two models discussed above. The key question is whether the DNA polymerase 5 spectrum is substantially the same in both URA3 orientations, as predicted by the first model, or essential- ly different, as predicted by the second model. A much larger sample of mutations will be required to answer this question.

Acknowledgements. We thank Alan B. Clark and Dinh C. Nguyen. for sequencing FOA R mutant DNAs. This work was partially sup- ported by the International Scientific Research Program and Grant- in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan to A.S.

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