polina shcherbakova and youri i. pavlov - genetics · and pavlov 1993). ura+ strains were...

3' + 5' Exonucleases of DNA Polymerases E and 6 Correct Base Analog Induced DNA Replication Errors on Opposite DNA Strands in Saccharomyces cermkiae

Polina V. Shcherbakova and Youri I. Pavlov

Department of Genrtics, St. Petenburg Stale University, s1. Petrrsburg 199034, Russia Manuscript received July 1 1, 1995

Accepted for publication November 15, 1995

ABSTRACT The base analog 6-Nhydroxylaminopurine (HAP) induces bidirectional GC + AT and AT + GC

transitions that are enhanced in DNA polymerase E and 6 3' + 5' exonuclease-deficient yeast mutants, po12-4 and pol?-01, respectively. We have constructed a set of isogenic strains to determine whether the DNA polymerases 5 and E contribute equally to proofreading of replication errors provoked by HAP during leading and lagging strand DNA synthesis. Site-specific GC + AT and AT + GC transitions in a Pol+, po12-4 or pol?-01 genetic background were scored as reversions of ura3 missense alleles. At each site, reversion was increased in only one proofreading-deficient mutant, either po12-4 or pol?"Ol, depending on the DNA strand in which HAP incorporation presumably occurred. Measurement of the HAP-induced reversion frequency of the urn? alleles placed into chromosome ZZInear to the defined active replication origin ARS?OCi in two orientations indicated that DNA polymerases t and 6 correct HAP-induced DNA replication errors on opposite DNA strands.

A variety of prokaryotic and eukaryotic DNA polymerases possess an associated 3' + 5' exonuclease

that removes incorrectly inserted nucleotides during DNA synthesis, providing an important step in main- taining high fidelity of genome replication (ECHOLS and GOODMAN 1991; WANG 1991). In the yeast Saccharo- myces cerevisiae, three DNA polymerases are essential for chromosomal DNA replication, DNA polymerases a, b and E (SUGINO 1995). The precise role of each polymerase at a replication fork is unknown. Current models suggest that DNA polymerase a-primase complex initi- ates DNA synthesis at replication origins and on the lagging strand while DNA polymerases S and E function during elongation. According to one model (MORRISON et al. 1990), DNA polymerase E synthesizes the leading strand of DNA and DNA polymerase S completes the synthesis of Okazaki fragments on the lagging strand. Another model suggests that DNA polymerase E synthesizes Okazaki fragments and DNA polymerase S is re- sponsible for the leading strand synthesis (BURGERS et al. 1991). However, in vitro replication of DNA from the SV40 origin requires only DNA polymerases a and S suggesting that DNA polymerase E might play an essential role in replication-linked DNA repair (WAGA and STIILMAN 1994).

DNA polymerases b and E possess an associated 3' +

5' exonuclease proofreading activity that has been shown to participate in correcting errors of DNA replication in vivo (MORRISON et al. 1991, 1993; SIMON et al. 1991). In the present study, we address a question

Cmr~.~,ondingauthorrY. 1. Pavlov, Department ofcenetics, St. Peters- burg State University, Universitetqkaya emb. 7/Y, St. I'etersburg, 199034 Russia. E-mail: p a v l ~ ~ ~ p h g . b r i . p u . r ~ l

(knetics 1 4 2 717-726 ( M a I < h , 191Ki)

whether the 3' + 5' exonucleases of DNA polymerases S and E contribute equally to proofreading of replication errors induced by the base analog 6-Nhydroxyl- aminopurine ( H A P ) during either leading or lagging DNA strand synthesis.

HAP is highly mutagenic in growing yeast cultures (SORENSON el al. 1981; PAvLOV et al. 1991). Most of the genetic and biochemical evidence suggests that HAP provokes mutations during DNA synthesis due to its ambivalent pairing capacity. dHAP-triphosphate replaces both dATP and dGTP during in vitro DNA synthesis by prokaryotic and eukaryotic DNA polymerases (ABDuL-MASIH and BESSMAN 1986). Both dCTP and dTTP can be incorporated opposite HAP during in vitro replication of HAP-containing template oligonucleotide by Klenow fragment of Escherichia coli DNA polymerase I (Y. I. PAVLOV and T. A. KUNKEL, unpublished results). Template HAP can direct C incorporation at this site in vivo (NOSKOV rt al. 1994). Consistent with this ambiguous pairing, HAP induces both GC +

AT and AT + GC transitions in yeast (SHCHERBAKOVA and PAVLOV 1993; NOSKOV et al. 1994). Mutations that impair excision, mutagenic, recombinational o r mis- match repair do not affect HAP-induced mutagenesis, therefore HAP does not create DNA lesions that can be processed by these repair systems. Several mutations in the POL2 gene encoding the DNA polymerase t reduce HAP-induced mutagenesis while mutations in the conserved 3' "* 5' exonuclease site of DNA polymerases 6 and E increase it several-fold, indicating that HAP mutagenic action is mediated by the DNA replication apparatus and that a fraction of HAP- induced mispair events can be proofread by replicative DNA polymerases (P. V. SH(:HERBAKOVA, V. N.

718 P. V. Shcherbakova and Y. I. Pavlov

NOSKOV, M. R. PSHENICHNOV a n d Y. I. PAVLOV, unpublished observations).

In this work, we studied the effect of proofreading by DNA polymerases t and 6 on HAP-induced reversion of several uru3 missense alleles. The uru3 alleles used can revert via either GC + AT or AT -+ GC transitions provoked by HAP incorporation into either leading or lagging strand of DNA. Our results indicate that 3' +

5' exonucleases of the DNA polymerases 6 a n d t correct HAP-induced replication errors on opposite DNA strands.

MATERIALS AND METHODS

Media and chemicals: We used standard rich (YPD) and minimal (SD) media (SHERMAN et al. 1986). L-canavanine (Sigma) was added to SD medium at 40 mg/l for selection of canavanine-resistant (Canr) mutants. 5-fluoroorotic acid (FOA) containing medium (BOEKE et al. 1984) was used for selection of ura3 mutants. HAP was synthesized according to the method of GINER-SOROI.~A and BENDICH (1958). HAP was dissolved in dimethylsulfoxide (DMSO, Sigma) before adding to YPD. Ethylmethanesulfonate (EMS) was from Sigma.

Strains and plasmids: S. cereuisiue haploid strains CG379 ( MATa a d d - l trpl-289 his 7-2 leu2-3,112 uru3-52) and XAM8A (same but bearing the po12-4 mutation, which impairs 3' -+ 5' exonuclease activity of DNA polymerase E ) (MORRISON et al. 1991) were kindly provided by A. MORRISON. L1-CG379 and Ll-XAM8A are derivatives of CG379 and XAMBA with the insertion of bacterial transposon Tn5 in the LYSP gene constructed as described (GORDENIN et al. 1991). The pol3-01 mutation leading to 3' + 5' exonuclease deficiency of DNA polymerase 6 was created in the chromosome of L1-CG379 by integration and excision of plasmid YIpAM26 as described (MORRISON et al. 1993). Presence of the $013-01 mutation was indicated by an -100-fold increase in both the frequency of spontaneous mutations to canavanine resistance and the reversion of his7-2. We introduced ura3-24, ura3-29, ura3-4 and ura3-2 missense mutations ( SHCHERBAKOVA and PAVI.OV 1993) into chromosome of L1-CG379 and its po12-4 and pol?- 01 derivatives by a two-step procedure. First, the uru3-52 allele of each strain [which is a Ty insertion in the 5' part of the URA3 coding region (ROSE and WINSTON 1984)] was con- verted to wild type (URA3) by transformation with the BglII- StuI fragment from pFL34 containing the 5' half of the URA3 gene. Southern blot hybridization showed the presence of only the wild-type URA3 allele in the transformants. The chromosomal URA3 gene was then replaced by different ura3 missense alleles using plasmids containing the LEU2 gene and one of the ura3 alleles cloned into pUC18 (SHCHERBAKOVA and PAVLOV 1993). Ura+ strains were transformed with plasmids digested with NcoI (which cuts inside the sequence of the ura3 allele) to ensure integration into the URA3 locus. Leu+ Ura+ transformants were plated onto 5-fluoroorotic acid (FOA) containing medium to select for excision of the plasmid with the wild-type URA3 gene. Excision events give rise to Ura- Leu- auxotrophs that can be easily distinguished from Ura- Leu' colonies resulting from gene conversion. For each strain the presence of the ura3 missense mutation was confirmed by DNA sequence analysis.

To insert ura3 missense alleles into chromosome ZZI near to the ARS306 replication origin (NEWLON et al. 1991), Urd+ derivatives of L1-CG379 and isogenic po12-4 and pol3-01 mutant strains were modified by the deletion of the entire URA3 gene in chromosome Vusing plasmid pJL164 (constructed by J. LI and kindly provided to us by D. CORDENIN). The URA3

gene was then inserted in both orientations -4.4 kb distal to the ARS306 replication origin using two variants of plasmid PAM92 (MORRISON et al. 1993, 1994). Correct chromosomal position of the Ura+ integrants was confirmed by Southern blot hybridization. The wild-type URA3 gene in chromosome Illwas then replaced by the ura3 missense alleles as described for the UR43 locus in chromosome V.

Measurement of revertant frequency: To measure the frequency of spontaneous and HAP-induced reversion, yeast cultures were started for each determination from -10' cells per milliliter in liquid W D medium containing control solvent or HAP solution. Cells were grown to stationary phase at 30" and plated after appropriate dilutions on selective medium lacking uracil and on YPD medium to determine revertant and viable counts. Aliquots of appropriately diluted cells were plated on medium containing 40 mg/l L-canavanine. The frequency of canavanine-resistant (Canr) mutants was measured in each experiment to verify that all isogenic proofreading-proficient strains responded similarly at each HAP dose used and the proofreading-deficient mutants had increased HAP-induced Can' mutant frequencies as expected from the early data (P. V. SHCHERBAKOVA, V. N. NOSKOV, M. R. PSHE- NICHNOV and Y. 1. PAvLOV, unpublished data). W D plates were incubated for three days at 30" before counting. For revertant or Can' mutant counts, plates were incubated for 6 days. Revertant and Can' mutant frequencies were calculated by dividing the revertant or Can' mutant count by viable cell count.

To determine the frequency of EMSinduced reversion, stationary phase cells grown in liquid YPD medium were collected by centrifugation and suspended in sterile water at densities of 10' cells per ml. EMS was added to a final concen- tration of 2%. Cells were incubated with mutagen for 1 hr, collected by centrifugation, washed and plated on medium without uracil and on YPD medium for revertant and viable counts. To measure the UV-induced reversion frequency, appropriate dilutions of stationary phase cells were spread on selective medium and on YPD medium and plates were UV irradiated (60 J/m') before incubation.

The significance of differences between mutant frequencies was estimated using Wilcoxon-Mann-Whitney nonparametric criterion.

Other methods: Yeast genetic transformation was performed according to I r o et al. (1983). Blot hybridization was according to SAMBROOK et nl. (1989). Amplification of URA3 DNA by PCR and DNA sequencing were as described (SHCHERRA- KOVA and PAVI.OV 1993).

RESULTS

Experimental system for analysis of site-specific reversions: A set of isogenic yeast strains carrying differ- en t uru3 missense mutations (Table 1 ) in a Pol', PoZ2- 4 or pol3-01 genetic background was constructed as described in the MATERIALS AND METHODS. It has been shown previously that H A P induces GC -+ AT and AT + GC transitions in yeast. Therefore we used two uru3 alleles, uru3-24 and uru3-29, which can revert via GC + AT transitions mediated by HAP incorporation into the sense (uru3-24) or antisense (uru3-29) DNA strand, and two alleles, uru3-4 a n d uru3-2, which can revert via AT + GC transitions mediated by HAP incorporation into sense (uru3-2) or antisense (uru3-4) DNA strand. Pathways for HAP-induced reversion of the four urdalleles are illustrated in Figure 1. To check whether

Strand-Specific DNA Proofreading 719

TABLE 1

urn3 missense mutations used in this study

ura3 Mutant DNA Reversion Amino acid allele sequence" specificity change

278

257 ura3-24 AGA AGA TIT G + A Arg + Lys

uru3-29 AAT TCT IITA C + T Ser + Phe C +Ab Ser + Tyr

ura3-4 ACA CTC GGT T + C Leu + Pro

ura3-2 GAA &4C AGA A - t G Asn + Asp

605

271

a The sequence is of the coding DNA strand. The mutated base is underlined. Numbers above the sequences indicate position of the mutated base.

C257 + A substitutions do not restore the wild-type URA3 sequence but produce Ura+ revertants having the same growth rate on medium lacking uracil as do true revertants.

only specific transitions that restore the wild-type URA3 sequence can be scored as Ura+ revertants, we sequenced the URA3 gene from 91 independent Ura+ revertants, including spontaneous and HAP-induced revertants, for each uru3 allele obtained in Pol', po12-4 and pol3-01 strains. Only true revertants of uru3-24, uru3-4 and uru3-2 were found (24, 21 and 12 revertants analyzed, respectively). In the case of uru3-29, two classes of revertants were revealed: true revertants resulting from CG + TA transitions and another class resulting from CG + AT transversions at the same site (see Table 1) . Both types of reversion occurred spontaneously at comparable frequencies but HAP enhanced only transitions (data not shown).

Effect of pol24 and po13-01 mutations on site-specific reversions: Spontaneous and HAP-induced revertant frequencies for the four uru? alleles introduced into

A.

B.

C.

chromosome Vof the wild-type strain and proofreading- deficient po12-4 and pol?-01 mutants are given in Table 2. All four uru3 alleles revert spontaneously at frequencies of the order of lo-' in the Pol+ strain. Consistently with the reported spontaneous mutator phenotype of po12-4 and $1013-01 mutants (MORRISON et ul. 1991, 1993; SIMON et uZ. 1991), we observed increased spontaneous reversion frequencies in proofreadingdeficient strains. HAP efficiently induced both types of transition at the doses used. Analysis of the effects of po12-4 and pol?- 01 on HAP-induced revertant frequency revealed two classes of uru3 alleles. The frequency of "induced reversion of uru3-24 and uru3-4 was elevated in the $1012- 4 proofreading-deficient mutant while no increase of these reversions was observed in the po13-01 mutant. Relative (to wild type) revertant frequencies calculated as described in the legend to Table 2 were 3.7-4.1 for po12-4 and 0.6-0.9 for pol3-01 mutant. In contrast, the frequency of uru3-29 and uru3-2 reversion was increased only in the poZ3-01 mutant, which displayed relative revertant frequencies from 3.6 to 8.4 for these two alleles while po12-4 had no significant effect. (Note that relative frequencies are only minimal estimates in the case of uru3-29as the value of spontaneous reversion frequency in exonuclease-deficient strains was composed of both transition and transversion frequencies; see above.) The only exception was the reversion of uru3-2 at the HAP dose of 50 ,ug/ml. In this case the po12-4 mutant also had a slightly increased reversion frequency. The 2.2- fold difference was statistically significant ( P < 0.01).

Increased reversion frequency in a proofreading-deficient mutant is expected if replication errors leading to the reversion are corrected by the corresponding DNA polymerase in wild-type cells. Therefore we con- cluded that in each particular reversion pathway HAP- induced replication errors are preferentially corrected by only one DNA polymerase, either DNA polymerase

ura3-29 ura3-24 ura3-4 ura3-2

-c- " - T- A- + ~~~ G ~ +A"- - -- A" --T -

t C- -H* - T- -H* FIGURE 1 .-Pathways for HAP-in-

and thin arrows designate sense and antisense DNA strands of the URA3 gene, respectively. H, HAP-containing nucleotide in DNA. (A) HAP incorpo-

+ H" ."-". ~~ t H-- - t --T- duced reversion of ura3 alleles. Thick

i I I 1

T* - H- - C* H- ration. (B) replication on HAP-con- +. c ~~~ taining template. (c) reversion fixa-

i tion.

720 P. V. Shcherbakova and Y. I . Pavlov

TABLE 2 "induced revertant frequencies for wild type and proofreading-deficient mutants

ura? Reversion DNA polymerase allele we mutation Spontaneous 50 pg/ml HAP 100 pg/ml HAP

Revertants per lo7 survivors

ura?-24 GC + AT p012-4 -

Pol?-01 ura329 CG + TA -

p01.2-4 Pol?-01

urn?-4 TA -+ CG POL?-4 -

Pol?-01 ura3-2 AT + GC -

p012-4 Pol?-01

<0.2 0.8 0.3 0.2 8

21 <0.2 <0.2

1.2 <0.1 <0.1 <0.1

182 t 14 (1.0) 722 t 119 (4.0) 147 t 21 (0.8) 77 2 6 (1.0) 65 t 12 (0.7)

329 t 40 (4.0) 62 2 7 (1.0)

257 t 19 (4.1) 56 2 8 (0.9)

5 t 1 (1.0) 11 t 2 (2.2) 42 t 5 (8.4)

383 2 50 (1.0) 1520 t 236 (4.0) 224 ? 34 (0.6) 139 t 25 (1.0) 142 t 31 (1.0) 613 t 51 (4.3) 120 ? 19 (1.0) 438 t 53 (3.7) 97 t 21 (0.8) 20 t 3 (1.0) 24 t 3 (1.2) 72 t 14 (3.6)

~

Revertant frequencies were measured for strains with ura3 alleles in chromosome V. Spontaneous revertant frequencies are given as medians for eight independent cultures of each strain. HAP-induced revertant frequencies are means t SE for six independent cultures. Relative revertant frequencies (in parentheses) aref. = [fHAp(exo-) - f (exo-)]/[fHAp(exo+) -f.,,(exo+)] whereX,(exo-) andfp(exo+) are spontaneous revertant frequencies for the proofreadingdeficlent strain and the corresponding wild-type strain, respectively, and fHm (exo-) and f H M (exo+) are revertant frequencies for the same strains after H A P treatment.

~~ ~~

sp.

E or DNA polymerase 6. Moreover, reversions of uru3- 24 and uru3-29, representing the same GC + AT pathway (see Figure 1) result from HAP-induced replication errors corrected by different DNA polymerases: by DNA polymerase E in case of uru3-24 and by DNA polymerase 6 in case of uru3-29. Similarly, replication errors leading to AT + GC transitions are preferentially proofread by DNA polymerase S in the case of uru3-2 and by DNA polymerase E in the case of uru3-4. We proposed that susceptibility of a mispair to proofreading by one or another DNA polymerase is determined by the DNA strand in which HAP is presumably incorporated. Alter- natively, it might depend on DNA sequence context of a mispair rather than the DNA strand in which replication error occurs. We examined the effect of po12-4 and pol3-01 mutations on reversion of seven additional uru3 missense alleles. From the known nucleotide sequence of these alleles, we expected that they revert via either CG "f TA transition through H A P incorporation into the same strand as in the case of uru3-29 reversion (three alleles) or AT -+ GC transition through HAP incorporation into the same strand as in the case of uru3-2 (four alleles), though we did not confirm the reversion specificity of these seven alleles by sequence analysis. Revertant frequencies for all seven alleles were increased in the pol3-01 mutant but little or no effect of the po12-4 mutation was observed, a result similar to that obtained with uru3-29and uru3-2 (data not shown).

Effect of uru3 allele orientation: To demonstrate further that different DNA sequence context of uru? alleles is not the reason for the observed different responses of uru3 alleles to po12-4 and pol301 mutations, we inserted uru3-24, uru3-29, uru3-4 and uru3-2 alleles into chromosome ZZZin both orientations near to the defined active

replication origin ARS306. This provided an opportu- nity to study proofreading of the same error generated by either leading or lagging replication apparatus in the same DNA sequence context. It has been shown that AH306is active in >90% of cell cycles (DESHPANDE and NEWLON 1992; A. MORRISON, personal communica- tion). Reversion frequencies for the two orientations of uru3 alleles are given in Table 3 and the effect of orientation on proofreading by DNA polymerases E and S is illustrated in Figure 2.

The effect of $1012-4 and po13-01 mutations on reversion of uru3 alleles in the LR orientation (Am306 is 5' to the coding DNA strand of the URA3) was essentially the same as that observed for u r d alleles in their natu- ral location in chromosome V: reversion of uru3-24 and uru3-4 was increased in the $1012-4 mutant (Figure 2, A and C) and reversion of uru3-29 and u r d 2 was increased in the po13-01 mutant (Figure 2, B and D). The only difference was that in the case of urd-24, the effect of po12-4 was greater than that in chromosome V. This can be seen by the comparison of the maximal relative revertant frequency of 17.4 for po12-4 urd-24 (Table 3, LR orientation) us. corresponding value 4.0 for chromosome V (Table 2). Changing the orientation of uru? alleles with respect to AH306 reversed the effects of po12-4 and $1013-01 on reversion. In RL orientation, reversions of uru3-24 and uru3-4 were increased by the p013- 01 mutation while the effects of the po12-4 mutation were barely detectable (Figure 2, A and C). Relative revertant frequencies were 3.2-18.7 for the $013-01 mutant and 1.7-2.7 for the po12-4 mutant (Table 3, RL orientation). In contrast, reversion of urd-29and uru2 2 in RL orientation was increased 3.7-36.7-fold in the po12-4 mutant (Figure 2, B and D; Table 3, RL orienta-

Strand-Specific DNA Proofreading

TABLE 3

Effect of uru3 allele orientation on proofreading by DNA polymerase E and 6

72 1

Revertants per 10' survivors

LR orientation DNA

lU orientation

uru? Reversion polymerase 50 p g / d 100 pg/ml 50 pg/ml 100 pg/ml allele type mutation Spontaneous HAP HAP Spontaneous HAP HAP

ura?-24 GC + AT - <42 '51 2 12 (1.0) '267 t 54 (1.0) <41 '7 2 2 (1.0) '30 t 6 (1.0) po12-4 '4 2 2 '873 2 183 (17.4) "3666 t 840 (13.8) <41 419 t 6 (2.7) '62 2 12 (2.1)

u r d 2 9 CG + TA - < '2 "32 2 5 (1.0) '119 -+ 11 (1.0) <"3 '160 Z 25 (1.0) "387 t 37 (1.0) po12-4 '30 2 14 '58 2 17 (0.9) '129 2 17 (0.8) '43 2 18 '686 2 26 (4.0) '1460 i 170 (3.7)

uru3-4 TA + CG - c 4 2 '50 2 8 (1.0) '141 -t 16 (1.0) "13 t 1 (1.0) "21 t 1 (1.0) po12-4 <'1 '248 ? 26 (5.0) '456 t 76 (3.2) <'l '22 t 6 (1.7) '40 ? 7 (1.9) pol?-01 '10 2 4 '60 2 13 (1.0) '129 -t 26 (0.8) < 32 '42 2 4 (3 .2) "168 t 35 (8.0)

ura?-2 AT -+ GC - <'l '19 2 5 (1.0) '61 ? 17 (1.0) <'1 "6 t 2 (1.0) "1 ? 2 (1.0) po12-4 <41 "29 t 8 (1.5) "64 2 10 (1.0) <41 '150 5 32 (25.0) '404 2 80 (36.7)

pol?-ol '9 t 5 "85 t 28 (1.5) '357 ? 39 (1.3) '' 4 2 1 '135 2 19 (18.7) '283 t 20 (9.3)

pol?-0l ' 68 2 26 '118 ? 14 (1.7) '539 ? 90 (4.0) '40 2 12 '228 t 39 (1.2) "320 i 35 (0.7)

Pol?-01 <'l "164 2 29 (8.6) 6460 t 85 (7.5) ' 4 ? 2 "49 2 9 (8.2) 'I23 -+ 24 (11.2)

uru? alleles were inserted into chromosome ZIInear the ARS?O6 replication origin in both orientations. LR and RL are the orientations in which ARS306 is 5' and 3' to the URA3 DNA coding strand, respectively. All frequencies are given as mean 2 SE. Superscripts indicate the numbers of independent determinations. Relative revertant frequencies calculated as described in the legend to Table 2 are in parentheses.

tion). pol3-01 had no significant effect on reversion of ura3-29 but increased reversion of ura3-2 up to 11.2- fold, although the effect of $1012-4 on reversion of ura3- 2 (36.7-fold increase) was greater than that of po13-01.

Relative revertant frequencies for the po12-4 mutant with a uru3 allele in one orientation usually did not differ significantly from those for the pol3-01 mutant with the same allele in opposite orientation. This suggests that overall efficiency of proofreading is similar for both LR and RL orientations of ura3 alleles. How- ever, in the wild-type strain having normal proofreading activity of both DNA polymerases, we observed up to 10-fold differences in reversion frequency between the two orientations of the same ura3 allele. These differences could reflect unequal frequency of DNA replication errors for the two orientations or, alternatively, some general orientation effect. To distinguish between these two possibilities, we studied the effect of orientation on reversion of ura3-24, uru3-29 and ura3-4 induced by alkylating agent ethylmethanesulfonate (EMS), which induces primarily GC + AT transitions in yeast (KOHALMI and KUNZ 1988), and by ultraviolet light (W), which induces transitions of both types (KUNZ et al. 1987; LEE et al. 1988). Figure 3 shows that E M S or UV-induced reversion did not depend on the orientation of the ura3 alleles, whereas HAP-induced revertants arose 5-10 times more frequently in the orientation in which proofreading defect of DNA polymerase E enhanced reversion.

DISCUSSION

In this study, we used HAP as a tool to investigate strand-specificity of proofreading by yeast DNA polymerases E and 6 in vivo. This approach was inspired

by the following observations. First, HAP induces DNA replication errors in uiuo at frequencies up to 200-fold above background (e.g., see PAVLOV et al. 1991). Second, in a reversion assay that scores site-specific transitions, the miscoding specificity of HAP (pairing with C or T) (ABDuL-MASIH and BESSMAN 1986; NOSKOV et al. 1994; Y. I. PAVLOV and T. A. KUNKEL, unpublished results) defines the DNA strand into which the HAP is incorporated. Third, HAP-induced DNA replication errors are subject to proofreading by both DNA polymerases E and S (P. V. SHCHERBAKOVA, V. N. NOSKOV, M. R. PSHENICH- NOV and Y. I. PAVLOV, unpublished observations). Therefore analysis of POL-4 and po13-01 effects on the induction of specific reversions can be used to identify the sites where proofreading is accomplished by DNA polymerases E and 6.

Similar to the classic model for base analog mutagenesis (FREESE 1959) the mechanism postulated for HAP- induced GC + AT transitions involves HAP incorporation opposite C during DNA synthesis and consequent T incorporation opposite HAP in the next cycle of replication (pathways for reversion of ura3-24 and ura3-29 are shown in Figure 1). Initial H A P incorporation opposite T and consequent C incorporation opposite HAP in the next replication cycle lead to AT -+ GC transition (see Figure 1 for pathways of reversion for uru3-4 and ura3-2). Exonucleolytic proofreading may eliminate mutagenic intermediates both at the H A P incorporation step and during DNA synthesis on HAP-containing templates. Thus while the site of the HAP incorporation is known for each reversion pathway, the DNA strand in which the replication error is corrected is not defined unequivocally. Two possibilities should be considered. First, the HAP may be treated by the DNA polymerases


A

E 4000

2 3500 ' 5 3000 00% 2500

g" 1500 3 2000

2 1000

& o 500

wild type pol24 ~013-01

B

ARS306 +"""-"-

c: 280

.? 240 5 200

160

5 120 Y 5 80

40

g o

Am306

:. . I

wild type pol2-4 pol3-01

FIGURE 2.-HAP-induced DNA replication errors on leading and lagging strands are corrected by different DNA polymerases. Sites for HAP incorporation for ura3-24 (A), ura3-29 (B), tcra3-4 (C) and ura3-2 (D) reversion are shown. Left and right forks represent LR and RL orientations of the ura3 allele, respectively, as indicated by open arrows. Below the forks, HAP-induced revertant frequencies for the corresponding urajalleles are diagrammed (data from Table 3; spontaneous revertant frequencies subtracted from frequencies measured at HAP dose of 100 pg/ml).

C

. .

D

ura3-4 < '7 - - "" I

RL

500 I I I I

e ? 400

8 400

'$ 300 3 & 200

>

DO- 300 0

a0

0 200

.$ 100 8 100 >

J o Y J o

wild type pol?-4 poll-01 wild type pol?-4 poU-01

724 P. V. Shchcrbakova and Y. 1. Pavlov

A.

B.

g 600 n

HAP EMS uv

1200 -

1000

800

600

400

200

0

I

i

HAP EMS uv

C. 150

HAP uv FIGITRE: X-Effect of urn3 allele orientation on HAP-, E M S

and UV-induced reversion of urn3-24 (A), urn3-29 (B) and 1~rn3-4 ( C ) in proofreading-proficient strains. Values are means for six independent determinations. Light and dark bars represent LR and RL orientations, respectively.

as the adenine analog and the HAP:C pair may be preferentially corrected by proofreading exonucleases. Al- ternatively, DNA polymerases may detect HAP as the guanine analog rather than the adenine analog and correct HAP:T pair.

If the HAP:C is preferentially proofread, then the effect of po12-4 and po13-01 mutations may be attributed primarily to proofreading during HAP incorporation in the case of GC -+ AT transitions, and to proofreading during replication of HAP-containing DNA in the case of AT -+ GC transitions. During reversion of ztrn3-24, HAP is incorporated opposite C by the leading strand DNA replication apparatus when urn3-24 is in the LR orientation and by the lagging strand DNA replication apparatus when urn3-24 is in the RL orientation (Figure 2, A). As indicated by the effects of po12-4 and @d3-01 on this reversion, proofreading of the HAP:C pair is

accomplished by DNA polymerase E in LR orientation of urn3-24 and by DNA polymerase 6 in the RL orientation of ztm3-24. I t is evident that in the case of reversion of zirf~?-29, HAP is incorporated during lagging strand synthesis and proofread by DNA polymerase b in the LR orientation and incorporated during leading strand synthesis and proofread by DNA polymerase E in the RL orientation (Figure 2R). During reversion of um3- 4 in the LR orientation, HAP is incorporated opposite T by lagging strand DNA synthesis (Figure 2C, LR orientation), but we detect proofreading of the HAP:C pair during leading strand synthesis when C is incorporated opposite the template HAP. This HAP:C pair is proofread by DNA polymerase E . In the RL orientation of z~rn3-4, HAP is incorporated into leading DNA strand but proofreading is accomplished during laggingstrand synthesis by DNA polymerase 6 (Figure 2C, RL orientation). Using the same logic, in the case of reversion of urn3-2, the HAP:C pair is corrected by DNA polymerase b during lagging strand synthesis when urn3-2 is in the LR orientation and by DNA polymerase E during leading strand synthesis when ura3-2 is in the RL orientation (Figure 2D). Thus, if HAP:C is preferentially proofread, the DNA polymerases E and 6 may be identified as leading and lagging strand proofreading enzymes, respectively. The opposite assumption, that HAP:T pair is preferentially corrected by the proofreading exonucleases, would define DNA polymerase 6 as the leading strand proofreading polymerase and DNA polymerase E as the lagging strand proofreading polymerase.

At present there is no data on the relative efficiency of proofreading of the W : C and HAP:T mispairs that would allow to distinguish behveen these two possibilities. It has been shown that HAP exists in aqueous solution predominantly in the amino form (see MOROZO\~ P/

nl. 1982), which can pair with T forming two hydrogen bonds, similar to a normal A T pair. The imino tauto- mer fraction is -30% (E. BUDOWSKI, personal commu- nication). HAP in imino form can pair with C via hvo hydrogen bonds, thus forming a less stable pair than normal G:C pairing. It is possible that DNA polymerases could discriminate against the HAP:C pair both at the base selection and the proofreading step to a much greater extent than against the HAP:T pair. Consistent with this proposal, dHAPTP replaced dATP more efficiently than dGTP in in d r o DNA synthesis with DNA polymerase I or DNA polymerase I11 of E. coli and with T4 DNA polymerase (ARDL~I,-MASIH and BESSMAN 1986) while T was more efficiently incorporated opposite template HAP than C by exonuclease-deficient Klenow fragment of DNA polymerase I of I<. coli Cy. I. PA\l.O\' and T. A. KUNKEI., unpublished results). Therefore at the incorporation step the HAP:T pair is preferred over the HAP:C pair. This is more easily reconciled with the assumption that the HAP:C pair is a more probable substrate for proofreading exonucleases than the HAP:T pair and, therefore, the DNA polymerases E and

Strand-Specific DNA Proofreading 725

S proofread DNA replication errors on the leading and lagging DNA strands respectively. Further studying of the HAP base pairing specificity and proofreading of HAP-containing pairs is necessary to define which of the two polymerases is the leading and which is the lagging strand major proofreading polymerase.

To date, only one attempt has been undertaken to assign specific roles to DNA polymerases E and 6 in proofreading of DNA replication errors. Spectra of spontaneous mutations at the URA? gene placed in two orientations relative to the ARS?06 origin in $1012-4 and pol30I strains were compared. Reversal of the orientation of URA? did not affect the spontaneous URA? mutation rates of po12-4 and pol30I strains but did change the spectrum of spontaneous mutations generated in both proofreadingdeficient mutants, indicating that DNA polymerases E and S can correct DNA replication errors on opposite strands (MORRISON and SUCINO 1994). However the number of mutations sequenced in that study was insufficient to estimate whether the mutational spectra in the two orientations differed significantly.

The involvement of the DNA polymerases E and S in proofreading on opposite DNA strands has relevance to the organization of multisubunit protein complex at a replication fork. Current models for eukaryotic DNA replication are based mostly on the studies of biochemical properties of DNA polymerases. DNA polymerase E

was proposed to be the leading strand polymerase as it is the most processive enzyme among eukaryotic DNA polymerases (MORRISON et al. 1990). On the other hand, it was shown that proliferating cell nuclear antigen (PCNA) is required for leading strand synthesis (PRELICH et al. 1987). Because PCNA increased the pro- cessivity of purified DNA polymerase 6 (TAN et al. 1986), it was proposed that this DNA polymerase synthesizes the leading DNA strand (BURGERS et al. 1991). Later it was shown that both 6 and E require PCNA for processive synthesis (PODUST et al. 1992). Replication of plasmid DNA containing the SV40 origin can be recon- stituted in vitro with only DNA polymerases a and S (WAGA and STILLMAN 1994) while three DNA polymerases, DNA polymerases a , 6 and E are essential for viabil- ity in yeast. The only indirect genetic evidence for specific roles of DNA polymerases E and 6 for in vivo chromosomal DNA replication is the enhancing effect of DNA polymerase S mutation on Tn5 excision in yeast, which is believed to occur during lagging strand DNA replication (GORDENIN et al. 1992). If the strand-specificity of proofreading exonucleases reflects the strandspecificity of DNA synthesis by corresponding DNA polymerases, we can regard our results as a proof that DNA polymerases E and S replicate opposite DNA strands in yeast. This proposal is only tentative as proofreading activity functioning on a DNA strand may not be related to the DNA polymerase that accomplishes DNA synthesis on a given strand. For example, DNA

replication errors generated by DNA polymerase (Y

(which has no proofreading activity) can be corrected by the exonuclease associated with DNA polymerase 6 (PERRINO and LOEB 1990), and DNA polymerases E and S can compete for the same pool of replication errors (MORRISON and SUGINO 1994). Work is in progress to reveal the effects of mutations in the polymerase dc- mains of DNA polymerases E and S on strand-specific replication errors.

Although “the main proofreading DNA polymerase” could be clearly identified for each orientation of each ura? allele, a slight effect for the second DNA polymerase was sometimes revealed (“second polymerase effect”). Usually it was no more than twofold, but, in the case of ura?-2 in the RL orientation, the effect of pol?- 01 was as great as 11.2-fold. This may reflect a small level of proofreading of the HAP:T pair (assuming that HAP:C pair is preferentially proofread). Proofreading during HAP incorporation opposite T would decrease the frequency of AT + GC transitions while the frequency of GC + AT transitions would not be significantly affected by proofreading of T incorporated opposite template HAP as T again would be favored for incorporation in this site. The effect of the second polymerase can be also explained if we propose that in a small fraction of replicative cycles ARS?06 fails to initiate replication and the URA3 gene is then replicated by the fork moving in the opposite direction from ARS305, which is -40 kb distal to the ARS306 (NEWLON et al. 1991). Then the DNA strand that is the leading strand most of the time would be replicated by lagging strand replication apparatus in a small fraction of replication events. The fraction of replication events in which ARS?06 fails to initiate replication was not estimated precisely though it is apparently <lo% (DESHPANDE and NEWLON 1992). For the two other highly active replication origins ARS?07 and ARS?09 this fraction is as small as 0.3% (DERSHOWITZ and NEWLON 1993). Finally, one more explanation for the “second DNA polymerase effect” comes from the observation that 3‘+ 5’ exonucleases of the DNA polymerases E and S can act competitively (MORRISON and SUCINO 1994). This suggests the possibility that each DNA polymerase correcting replication errors preferentially on its own strand can also correct some fraction of errors on the opposite strand. All three mechanisms could potentially contribute to the observed “second polymerase effect” and the relative contribution of each mechanism may be site specific.

For three of the four ura? alleles, we observed that HAP-induced reversions in proofreading-proficient strain were up to 10 times more frequent in the orientation in which DNA polymerase E proofreads HAP-induced errors (Figure 3). On the assumption that a polymerase preferentially corrects its own errors, DNA polymerase E appears to be more error-prone than 6 in respect to HAP-induced errors. This is consistent with


two observations. First, mutations affecting the polymerase domain of DNA polymerase E greatly reduced HAP- induced mutagenesis (P. V. SHCHERBAKOVA, V. N. NOSKOV, M. R. PSHENICHNOV, and Y. I. PAVLOV, unpublished data). Second, 92% of the HAP-induced forward mutations in the URA3 gene can be ascribed to replication errors in one DNA strand (SHCHERBAKOVA and PAVLOV 1993). This asymmetry can be explained if the two DNA polymerases replicating opposite DNA strands have different fidelity in respect to HAP-induced replication errors. Alternatively, the frequency of these errors during the leading and lagging strand synthesis may be unequal regardless of whether they are synthesized by the same polymerase or by two different polymerases.

We would like to thank Dr. A. MORRISON for the yeast strains and plasmids, Dr. D. GORDENIN for the pJL164 , and Drs. A. MORRISON, A. SUGINO, T. A. KUNKEL and B. A. KUNZ for helpful discussions. We thank Dr. C. BENNET for critically reading the manuscript. The research described in this publication was made possible in part by grant JDHl00 from the International Science Foundation and the Russian Government to Y.I.P. This research was also supported in part by the Russian program, Frontiers in Genetics grants 12-5 and 25 and Russian Fund for Fundamental Scientific Research grant 95- 041 1583a.

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polina shcherbakova and youri i. pavlov - genetics · and pavlov 1993). ura+ strains were...

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