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Molecular and Cellular Biology, March 1999, p. 2000-2007, Vol. 19, No. 3
Chromosome Stability Group, Laboratory of
Molecular Genetics, National Institute of Environmental Health
Sciences, Research Triangle Park, North Carolina
27709,1 and Department of Genetics, St.
Petersburg State University, St. Petersburg 19034, Russia2
Received 1 July 1998/Returned for modification 5 August
1998/Accepted 18 November 1998
Replication fidelity is controlled by DNA polymerase proofreading
and postreplication mismatch repair. We have genetically characterized
the roles of the 5' Chromosome replication fidelity is
generally considered to be determined by a combination of base
selection and error correction activities of DNA polymerases along with
postreplication mismatch repair (MMR). The combined effect in
Escherichia coli results in an overall error rate that is as
low as 10 In eukaryotes the three polymerases required for chromosome replication
are polymerase The postreplication MMR system is responsible for the correction of
errors generated during replication. Based on in vitro analysis with
purified proteins, the steps in E. coli MMR include mismatch
recognition by MutS and MutL proteins, methyl-directed strand
discrimination, incision by the MutH endonuclease in the unmethylated
nascent strand at sites opposite methylated GATC, degradation from a
nick towards the mismatched site by exonucleases (ExoI, ExoVII, or
RecJ), and gap filling by polymerase III holoenzyme followed by DNA
ligation (21).
Except for mismatch recognition, relatively little is known about the
MMR mechanism in eukaryotes. Many genes homologous to E. coli
mutS and mutL have been cloned and studied
(21). Strand-specific MMR was demonstrated in human cell
extracts when a single-strand nick was introduced on either side of a
mismatch (7, 37), suggesting that both 5' We have examined the role of various exonucleases in MMR as part of our
efforts to screen and characterize MMR genes in the yeast S. cerevisiae. For example, the small mutator effect of an
exo1 mutation as compared to that of an msh2
mutation suggests that there are additional exonucleases involved in
MMR. A synergistic mutator effect was found for defects in the Exo1
nuclease and the Pol General genetic and molecular methods.
Yeast standard media
(30) and yeast-extract-peptone-dextrose (YPD) medium with
G418 (45) have been described previously. Yeast cells were
grown at 30°C. Yeast transformations were performed according to the
methods of Gietz and Schiestl (10). The preparation of
bacterial media and general molecular methods have been described previously (25).
Strains and plasmids.
A series of isogenic strains were
constructed from the original CG379 (MAT
0270-7306/99
The 3'
5' Exonucleases of DNA Polymerases 
and
and the 5'3' Exonuclease Exo1 Have Major Roles in
Postreplication Mutation Avoidance in Saccharomyces
cerevisiae
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
3' Exo1 and the 3'5' DNA polymerase exonucleases in mismatch repair in the yeast Saccharomyces
cerevisiae by using various genetic backgrounds and highly
sensitive mutation detection systems that are based on long and short
homonucleotide runs. Genetic interactions were examined among DNA
polymerase 
(pol2-4) and 
(pol3-01)
mutants defective in 3'5' proofreading exonuclease, mutants
defective in the 5'3' exonuclease Exo1, and mismatch repair mutants
(msh2, msh3, or msh6). These three exonucleases play an important role in mutation avoidance.
Surprisingly, the mutation rate in an exo1 pol3-01 mutant
was comparable to that in an msh2 pol3-01 mutant,
suggesting that they participate directly in postreplication mismatch
repair as well as in other DNA metabolic processes.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
10 errors per replicated nucleotide
(27). In E. coli, the 
polymerase subunit of
DNA polymerase III holoenzyme, encoded by the dnaE gene,
determines base selection and the subunit (dnaQ)
provides proofreading. These subunits are tightly bound together with
the 
subunit to form the polymerase III core (16). The
errors left by the polymerase III holoenzyme are corrected by the
MutHLS MMR system (28).
, , and , encoded by the POL1,
POL3, and POL2 genes, respectively. Polymerase
(Pol) is responsible for the synthesis of primers for Okazaki
fragments in the lagging strand, and the and polymerases, it
has been proposed, are responsible for lagging and leading DNA strand
replication (35), although their specific roles have not
been established. Unlike Pol, the and polymerases also have
a 3'5' proofreading exonuclease activity in their N-terminal regions
(14, 22, 32). In the yeast Saccharomyces
cerevisiae, the point mutations pol3-01 and pol2-4, which eliminate the proofreading activities of the
and polymerases, respectively, result in a frameshift and base
substitution mutator phenotype (22, 23).
3' and 3'5'
exonucleases may be involved. The 5'3' exonuclease Exo1 of the yeast
S. cerevisiae has been implicated in MMR. The
EXO1 gene, which is homologous to the
Schizosaccharomyces pombe EXO1 gene (36), was
isolated in a two-hybrid interaction screen with yeast MSH2
(38). Tishkoff et al. (38) concluded that
EXO1 and MSH2 are in one epistasis group;
however, other interpretations are possible. In an
exo1-deficient S. cerevisiae strain, the
canavanine resistance (Canr) forward and hom3-10
reverse mutation (
1 frameshifts) rates are increased only eight- and
sixfold, respectively, in comparison with a wild-type strain. This is
much lower than the corresponding 25- and 850-fold increases observed
in msh2 mutants (38), and the msh2
exo1 double mutant exhibits rates comparable to that of the single
msh2 mutant. However, it cannot be ascertained from these
data whether MSH2 and EXO1 have an epistatic or
additive interaction (i.e., single versus separate pathways), since the rate in the msh2 mutant differs little from the sum of the
rates in the msh2 and exo1 mutants. Since both
Exo1 and Msh2 are involved in homologous recombination (9, 26,
34), their physical interaction could be related to this process
rather than MMR.
3'5' proofreading exonuclease, suggesting
that these activities participate in postreplication steps. Cells
deficient in both DNA Pol 3'5' proofreading exonuclease and
either Exo1, Msh2, or DNA Pol 3'5' proofreading exonuclease
(24) were inviable, implying a strong synergistic
interaction. On the other hand, diploid strains defective in both DNA
Pol 3'5' proofreading exonuclease and Exo1 or Msh2 were viable.
Surprisingly they exhibit comparable levels of hypermutability. We
propose that the Pol and Pol proofreading exonucleases as well as
Exo1 may play a major role in mutation prevention.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
ade5-1 his7-2
leu2-3,112 trp1-289 ura3-52) (22) and
pol2-4 and pol3-01 derivatives (40).
The pol2-4 and pol3-01 mutations are point
mutations in the exonuclease domain of the POL2 and
POL3 genes, respectively, resulting in the loss of 3'5' proofreading exonuclease activities in the corresponding polymerases (22-24). These strains contain modified insE
inserts in the chromosomal LYS2 gene, where the
A4 run was changed to A5, A12, or
A14 (40). Mutations of the following DNA
metabolism genes were introduced into these strains: MMR genes
MSH2, MSH3, and MSH6 and the 5'3' exonuclease gene EXO1.
Mutator screening with a disruption library. Plasmid DNA of a gene disruption library, kindly provided by M. Snyder (4), was digested with NotI to release yeast genomic DNA fragments containing Tn3::lacZ-LEU2 inserts and transformed into Pol+ lys2::insE-A14 or pol2-4 lys2::insE-A14 strains, and Leu+ transformants were selected. The transforming DNA fragments randomly knock out different nonessential genes through homologous recombination. The Leu+ transformants were replicated to complete medium without lysine in order to identify clones that could yield Lys+ papillae. These potential mutators were verified and crossed with tester strain 1036 (lys2-BX pms1 msh2 mlh1) to identify possible MMR mutators. The nature of the disrupted genes in the remaining clones was determined by using an inverse PCR technique instead of the rescue plasmid technique described by Burns et al. (4). First, genomic DNA from the clones was isolated and digested with the frequently cutting HpaII or TaqI endonucleases. These enzymes will cut inside the Tn3-lacZ-LEU2 insert as well as nearby regions. Digested DNA fragments were circularized by DNA ligation. After circularization, the junction region between the yeast genomic DNA and Tn3-lacZ-LEU2 insert was amplified with the following primers: lacZ, 5'-GCGGGCCTCTTCGCTATTACG-3', and lacZ-2, 5'-TGAATGGCGAATGGCGCTTTG-3'. The PCR products were sequenced with the lacZ-1 primer, 5'-GTCACGACGTTGTAAAACGACG-3'.
DNA and mutation analysis. An ABI sequencer was used for DNA sequencing. Mutation rates were determined by a fluctuation test by the method of the median (19) with at least 12 independent cultures. The nature of the Lys+ revertants was identified by sequencing the reversion window of the lys2::insE insert as described previously (41). The DNA regions sequenced between the Tn3-lacZ-LEU2 inserts and the yeast genome were identified by using the S. cerevisiae DNA database (26a), and Swissprot was used to search the protein database (24a).
Gene replacement and disruption. The following genes were disrupted: MSH2, MSH3, MSH6, and EXO1. For MSH2 disruption we used a SacI-PstI msh2::LEU2 fragment from p203 (39). The BamHI-AatII fragment from pmsh3::LEU2 (29) was used to disrupt the MSH3 gene.
The entire open reading frames of the EXO1 and MSH6 genes were deleted by the PCR disruption technique with the kanMX module (45) and primers described below. Lowercase letters indicate nucleotide sequences that belong to the kanMX cassette; DNA sequences belonging to the genes are written in uppercase. For the EXO1 gene we amplified the kanMX cassette with EXO1-kanMX-3' (5'-TTGGCTTGACTTAGTAGTTTCGATGTCCCTTTTCTTACTTatcgatgaattcgagctcg-3') and EXO1-kanMX-5' (5'-AGGTATGAAGGAGAAGTGTTAGCCATTGATGGCTATGCATcgtacgctgcaggtcgac-3'). For the MSH6 gene the following primers were used: MSH6-kanMX-5' (5'-CTACCCCTAA AACTTCTAAGACTGCACACTTCGAAAATGGatcgatgaattcgagctcg-3') and MSH6-kanMX-3' (5'-GTCCATCTCCGTACGCAATTCGAACGAAATCACTTTGTAAcgtacgctgcaggtcgac-3'). YPD medium with G418 was used for the selection of transformants (45). To verify the disruption of the EXO1 gene we used the following pair of primers for the EXO1 gene: EXO1-test-3 (5'-ATTGGGAAAGCAAGGAGATAG-3') and EXO1-test-5 (5'-TCTTCTTCCTCAGTTAAAGC-3'). The disruption of the MSH6 gene was verified by PCR with primers MSH6-test-5 (5'-CAGCTACCCCTAAAACTTC-3') and MSH6-test-3 (5'-TTCCAATCATAGTTCAAGACCCC-3'). The EXO1 gene was also disrupted with the HindIII-KpnI fragment (exo1::URA3) from plasmid p244. The p244 plasmid was constructed by first generating a PCR product of the chromosome EXO1 gene with primers EXO1-test-3 and EXO1-test-5. A BglII-NsiI fragment from the PCR product was cloned into the BamHI-PstI sites of the pUC19 plasmid, resulting in p243. The URA3 gene BglII-BglII fragment from pFL34 (3) was cloned into the BamHI site inside the EXO1 gene of p243, resulting in plasmid p244. To verify the disruption of MSH2 and MSH3 genes, PCR primers and conditions were used as described previously (42).Tetrad analysis.
The MAT pol3-01
lys2::insE-A4 and MAT pol3-01
lys2::insE-A5 strains were mated with strain
1036 (lys2-BX exo1::kanMX). In the diploid strains
the second copy of the EXO1 gene was disrupted with the
HindIII-KpnI
(exo1::URA3) fragment from p244. The diploid strains were sporulated, and tetrad analysis was performed. The spore
colonies with the wild-type POL3 gene were distinguished from pol3-01 mutants by using PCR. Genomic DNA from viable
spores was isolated and analyzed by PCR with two primers, p3
(5'-GGAGATACCAAATTACCA-3') (785-802) and d8
(5'-CTTGTACCATAAGCCTTC-3') (1512-1495). The PCR product
covers the pol3-01 mutation. The PCR products were digested with EcoRV; the pol3-01 mutation lacks an
EcoRV site (23).
Construction of homozygous diploid strains.
Haploid
leu2 strains were transformed with plasmid YEpHO (a gift
from Y. Chernoff) carrying the LEU2 marker and the
HO endonuclease gene. The HO endonuclease will
induce mating type switching in haploid strains from
MATa to MAT or vice versa. Haploid strains with the opposite mating types could form
MATa/MAT homozygous diploid strains.
Transformants with YEpHO were grown on YPD media to allow loss of the
plasmid. Single Leu clones were isolated. Diploid clones
were identified as nonmating with both MATa his3
and MAT his3 testers as well as giving a low forward
mutation rate to Canr due to the presence of two
CAN1 gene copies. For strains in which the LEU2
marker could not be used we utilized plasmid pGHO-TRP1 (1)
containing the TRP1 marker and the HO gene under
the GAL1-10 promoter. Mating type switching was induced in
galactose media for 8 h, and diploid clones were identified as
described above.
Plasmid loss rate measurement. To establish the requirement for the POL3 gene in strains with pol3-01 msh2 or pol3-01 exo1 combinations, the rate of loss of the plasmid-borne POL3 gene on pBL304 was determined. By using the URA3 marker on pBL304, the plasmid loss rate was determined from the median in a fluctuation test of 10 independent clones on 5-FOA (5-fluoro-orotic acid) media (2). For each fluctuation test, 10 5-FOA-resistant clones were analyzed by PCR to determine the loss of the POL3 gene as described above (see the paragraph on tetrad analysis).
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Isolation of mutators for a long A14 homonucleotide run. The MMR process is conserved in E. coli and eukaryotes. There are several E. coli MutS and MutL homologs that have been identified in yeast (21). While genes involved in mismatch recognition are well characterized, genes involved in later MMR steps such as strand discrimination, mismatch excision, and DNA resynthesis remain to be identified in S. cerevisiae. Previously a lys2::insE-A14 mutation system based on a homonucleotide run of 14 A that is hypersensitive to defects in MMR has been described (40). For example, an msh2 mutant exhibits a 10,000-fold increase in the Lys+ reversion rate over the wild type. Thus, even a relatively small impact of mutators can be detected. Using this sensitive system we tried to identify additional mutators affecting the instability of a long homonucleotide run through a saturation inactivation screen of nonessential genes.
The Pol+ lys2::insE-A14 (S1-A14) or pol2-4 lys2::insE-A14 (S3-A14) mutator detection strain containing the homonucleotide run A14 in the LYS2 gene was transformed by a gene disruption library (4), and colonies that exhibited higher Lys+ reversion rates were identified. As shown below, the pol2-4 background increases the magnitude of a mutator effect. Among more than 100,000 transformants of the Pol+ strain and 50,000 transformants of the pol2-4 mutant, 44 and 25, respectively, exhibited markedly increased Lys+ reversion frequencies. Of these hypermutable transformants, 34 and 19, respectively, carried mutations in the MMR gene PMS1, MSH2, or MLH1. The frequency of repeat mutants indicates that we have efficiently inactivated most nonessential genes that were likely to lead to an increase of the mutation rate in a sensitive system. Included in the remaining 10 hypermutable transformants of the Pol+ strain were 5 exo1-dhs1 mutants (ORF-YOR033C; the DHS1 gene also has been referred to as EXO1 [38]). The Tn3::lacZ-LEU2 inserts were at nucleotide positions 13, 104, 131, 150, and 163 from the EXO1 start codon. Among six hypermutable transformants of pol2-4, two were exo1 mutants and three were msh6 mutants. To establish that the increased reversion rate of the lys2::insE-A14 allele in the exo1 mutant strains was due to the disruption of the EXO1 gene and not to additional mutations elsewhere in the genome, the EXO1 gene was deleted from the S1-14A strain by using a kanMX cassette in combination with PCR (reference 45; see Materials and Methods). The disruption of EXO1 led to a 100-fold increase in the Lys+ reversion rate that was due to mutations in the A14 run (Table 1). In the six mutator clones lacking alterations in EXO1 or MMR genes (five from the Pol+ strain and one from the pol2-4 strain), disruptions were identified in the OXA1, CSD3, PMR2, ENA2, and 25S rRNA genes. Since the direct disruption of these genes (except 25S rRNA) in the S1-14A strain did not lead to a mutator phenotype, the mutator phenotype of these transformant clones is likely to be due to secondary mutator mutations elsewhere in the genome.
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Mutator activity of an exo1 mutant.
Based on
genetic and in vitro observations, exonucleases play an important role
in mutation avoidance in E. coli (5, 18, 44). We,
therefore, examined the role of EXO1 by using several assays
that reveal 1 and +1 frameshift mutations in or near short (A4 and A5) and long (A14 and
A12) homonucleotide runs within the
lys2::insE sequence (40). Forward
mutation to canavanine resistance was also examined in these strains
(see reference 30). Many of the
lys2::insE-A4 and
lys2::insE-A5 revertants were not due
to frameshift mutations in homonucleotide A4 and
A5 runs, respectively (Table 1). Instead, most mutations
were frameshifts located elsewhere in the reversion window (i.e., the
small region where mutations occur; see references
40 and 42). As expected, all
lys2::insE-A14 and
lys2::insE-A12 revertants were due to
1 and +1 nucleotide frameshift mutations in the A14 and
A12 runs, respectively (Table 1). Forward mutation rates in
the CAN1 gene were determined, but Canr
mutations were not sequenced.
1
frameshift mutations; there was only a twofold increase in +1
insertions in the A12 run (Table 1). The preference of the mutator effect for deletions over insertions is characteristic of
defects in MMR genes such as MSH2, MSH3, and
MSH6 (references 31 and
33; also see Table 1). The pattern of a relatively weak effect on short runs and a strong effect on long runs is typical
for an msh2 defect (reference 40 and
Table 1). These observations led us to determine the consequences of
combining these two mutations (i.e., exo1 and
msh2). Since there was no synergy for the combined
exo1 msh2 mutants as compared to msh2, it appears
either that msh2 is epistatic to exo1 or that
their effects are additive (Table 1). These modes of interaction cannot be distinguished because of the relatively small effect of the exo1 mutant on mutation rates.
Mutator phenotype resulting from a lack of DNA 3'5' Pol
proofreading and 5'3' Exo1 exonucleases.
The mutator phenotype
of the Exo1 defects suggests that this protein might be involved in
MMR. Since DNA polymerase proofreading and postreplication MMR act in
series, we examined the mutator phenotype of double mutants defective
in DNA Pol proofreading (pol2-4) and the 5'3'
exonuclease Exo1. The double mutant lacking proofreading and Exo1
activities exhibited a synergistic mutator effect for all mutation
rates examined (Tables 1 and 2). The pol2-4 mutant is a weak mutator (less than a sevenfold
increase over the wild type; Table 1). In the pol2-4 exo1
double mutant, mutation rates were increased by as much as 10- to
100-fold over those for either single mutant (Tables 1 and 2).
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5' proofreading exonuclease activity of DNA Pol
had been shown to have almost no effect on frameshift mutations in long
homonucleotide runs (
8 nucleotides) during replication
(40). The lack of proofreading in long homonucleotide runs
accounts for the absence of a multiplicative mutator effect when the
proofreading exonuclease mutant is combined with a complete (msh2) or partial (msh3 and msh6)
defect in the MMR system (Tables 1 and 2). The synergistic effect of
exo1 with the pol2-4 defect for mutations in long
homonucleotide runs suggests that the Pol-exonuclease can prevent
errors by a process other than replication proofreading (see Discussion).
Mutation in DNA Pol proofreading and Exo1 or Msh2 is
lethal.
The synergism of mutations in exo1 with
pol2-4 led us to examine the double mutant phenotype for
exo1 with pol3-01, a 3'5' exonuclease
deficient mutation of DNA Pol. The pol3-01 mutant exhibited a much stronger mutator effect than the pol2-4
strain in all assays examined (Table 1). We failed to obtain an
exo1 pol3-01 double mutant by the deletion of the
EXO1 gene in a pol3-01 strain. To analyze the
apparent synthetic lethality of the double mutant, we crossed a
pol3-01 and an exo1 mutant (see Materials and
Methods) and subsequently inactivated the second copy of the EXO1 gene with an exo1::URA3 DNA
fragment. Among 14 tetrads analyzed, all segregated as 2 viable:2
nonviable. Microscopic analysis after 4 days revealed that the two
spores that did not yield colonies underwent several divisions,
resulting in microcolonies containing approximately 100 cells. Using
PCR and restriction analysis (see Materials and Methods), we
established that the viable spores contained the wild-type copy of the
POL3 gene. Thus, exo1 pol3-01 mutants are inviable.
2 loss events per
cell/generation), the pol3-01 msh2 pBL304 or pol3-01
exo1 pBL304 strain demonstrated an undetectable level of plasmid
loss (5-FOAr colonies formed at a rate lower than
105 loss events per cell/generation). Based on PCR
analysis, the 5-FOAr clones derived from the pol3-01
msh2 pBL304 or pol3-01 exo1 pBL304 strain still retain
the wild-type copy of the POL3 gene. Thus, the pol3-01
msh2 and pol3-01 exo1 haploid double mutants are inviable.
Viability of msh2 pol3-01 and exo1 pol3-01
diploids.
A likely explanation for the inviability of the
pol3-01 msh2 and pol3-01 exo1 haploid strains is
that unedited replication errors generated by the
proofreading-deficient DNA Pol are lethal to a haploid cell. If this
were the case, then diploid strains might be viable due to the fact
that most mutations are recessive. We constructed diploid strains that
contained the pBL304 plasmid and had the following homozygous
mutations: pol3-01/pol3-01 msh2/msh2 and
pol3-01/pol3-01 exo1/exo1 (see Materials and Methods). The plasmid loss rates in the diploid strains were measured on 5-FOA media.
Unlike the haploid strain, all diploid strains were able to lose the
plasmid rapidly (3 × 103 loss
events/cell/generation).
Synergistic interaction between a DNA Pol 3'5' proofreading
defect and exo1 or msh2 mutations in diploid
strains.
The genetic interaction between pol3-01 and
msh2 or exo1 in diploid strains was studied by
examining the reversion of homozygous lys2::insE-A14 and his7-2
mutations. (While the his7-2 molecular defect is not known,
it has been used in several studies investigating proofreading and MMR
defects [22, 23, 24].) As shown in Table
3, the pol3-01/pol3-01
msh2/msh2 double mutant exhibits a His+ reversion rate
47-fold higher than that of the pol3-01/pol3-01 strain and
250-fold higher than that of the msh2/msh2 diploid. The rate
of Lys+ reversion in the A14 homonucleotide run
in the pol3-01/pol3-01 msh2/msh2 strain is similar to that
in an msh2/msh2 strain (Table 3). The data are consistent
with previous suggestions based on in vitro and in vivo data (17,
40) that DNA polymerase exonucleolytic proofreading becomes
ineffective during replication of long repetitive sequences (e.g., 8
nucleotides in a homonucleotide run). The strong synergistic
interaction between exo1 and pol3-01 was observed for Lys+ as well as His+ reversion (Table 3);
the reversion rates were multiplicative for the double versus single
mutants. The rate of Lys+ reversion for the A14
homonucleotide run in the pol3-01/pol3-01 exo1/exo1 strain
was 23-fold higher than the rate in the pol3-01/pol3-01 strain and 120-fold higher than the rate in an exo1/exo1
strain. The reversion rates to His+ and Lys+ in
the pol3-01/pol3-01 exo1/exo1 strain are approximately the same as those found in the pol3-01/pol3-01 msh2/msh2 double
mutant (Table 3). In contrast, in a Pol+ strain the
exo1 deficiency increases the mutation rate in the A14 run 100-fold less than an msh2 deficiency
(Table 1). Since DNA Pol exonucleolytic proofreading is inefficient
during replication of a long homonucleotide run (A14), the
synergistic effect between exo1 and pol3-01 for
mutations in long homonucleotide runs suggests that the
Pol-exonuclease can prevent errors by processes other than
replication proofreading.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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We have investigated mutator effects due to the inactivation of
the 5'3' Exo1 and 3'5' DNA polymerase exonuclease activities in
yeast. To investigate possible genetic networks of mutation avoidance,
we combined exo1 with mutations in MMR genes and/or deficiencies in the 3'5' proofreading activities of the DNA
polymerase genes. Based on the phenotypes of the mutants and double or
triple mutants, we propose that the exonuclease activities of Exo1,
Pol, and Pol participate directly in MMR.
Participation of the Exo1 5'3' exonuclease in MMR.
It has
been proposed that Exo1 may function in MMR (38). The
exo1 defect caused a nearly 100-fold increase in 1
frameshift mutations within the A14 homonucleotide run but
had a smaller effect in other assays (two- to threefold increases for
A4, A5, and A12 runs and a 6.5-fold
increase for the Canr forward mutation; see Table 1). In
all cases, the mutation rate in the exo1 msh2 mutant was
comparable to the rate in the msh2 mutant (Table 1 and
reference 38). This suggests that msh2 is
epistatic to exo1 and that Exo1 is involved in the
MSH2-dependent MMR pathway. (However, separate pathways
resulting in the additivity of mutation rates cannot be excluded
because the single exo1 mutant exhibits a low mutation
rate.)
3'5' proofreading exonuclease defect (pol3-01) results in
lethality for haploid strains, which is likely due to excessive errors. Diploid pol3-01 msh2 and pol3-01 exo1 strains are
viable and exhibit synergistic mutator effects (Table 3).
Participation of 3'5' exonucleases of Pol and Pol as well
as the 5'3' exonuclease Exo1 in error avoidance.
Proofreading
decreases with the increased size of the homonucleotide run that is
replicated (17, 40). Previously it has been shown that both
MMR and DNA Pol proofreading are efficient at preventing mutations
in short runs (synergy had been shown for pol2-4 and
pms1 or msh2 double mutants [24,
40], while only MMR prevents frameshift mutations in
homonucleotide runs larger than 7 nucleotides [40]).
It has been proposed that frameshift intermediates generated in long
runs escape Pol proofreading but are correctable by MMR. This would
explain the lack of synergy for mutations in long homonucleotide runs
when proofreading (pol2-4) and msh2,
msh3, or msh6 mutants are combined (Table 2 and
reference 40).
-exonuclease proofreading is absent in long homonucleotide runs,
we propose that this synergy is a manifestation of both gene products
(Exo1 and Pol) participating in error avoidance. While other
mechanisms might be involved we suggest that the Exo1 and Pol
exonucleases function in the MMR of long homonucleotide runs and
compete for substrates when mismatch recognition components are present
(i.e., Msh2, Msh3, and Msh6). Another possibility is that the MMR
system is saturated in a pol2-4 mutant due to the
accumulation of mismatches. Thus, a partial defect in MMR due to loss
of Exo1 might lead to a synergistic increase in the instability of long
homonucleotide runs. However, this contradicts the lack of synergy for
mutation of long homonucleotide runs in double mutants between
pol2-4 and either msh3 or msh6, which
are partially defective in MMR (Table 2).
The proposed role for the Exo1 and Pol exonucleases could occur if
the two types of exonucleases participate in the excision of mismatches
in a manner similar to that found for the RecJ and ExoVII 5'3'
exonucleases and the Exo1 3'5' exonuclease in E. coli MMR
(21). However, in E. coli the recJ
xseA double mutant defective in RecJ and ExoVII is not
hypermutable, implying that the system functions efficiently even when
restricted to a unidirectional mode of action (5). Recently,
Viswanathan and Lovett (44) reported that frameshift
mutations were stimulated in a RecJ Exo1
ExoVII triple mutant, although base substitution
mutations were not increased in several assays. This mutator effect was
primarily due to a synergistic interaction between the
Exo1 and ExoVII mutations.
Based on in vitro data, Longley et al. (20) have suggested
that DNA Pol participates in MMR at the resynthesis step. Our genetic data suggest that DNA Pol proofreading exonuclease may also
be directly involved in the excision step of MMR. Similar to the DNA
Pol proofreading exonuclease, DNA Pol-exonuclease also appears
inefficient during replication of the homonucleotide run
A14, so there is no synergistic interaction between
proofreading defects and msh2 (Tables 2 and 3). While the
haploid pol3-01 exo1 strain is inviable, the homozygous
diploid strain is viable and exhibits a strong synergistic mutator
interaction both for the instability of the A14 run and the
reversion of his7-2 (Table 3). The synergism between
exo1 and pol3-01 for mutations in the A14 run suggests that the Pol-exonuclease prevents
errors by processes other than proofreading. Since Exo1 is likely to be involved in MMR (reference 38 and this work) we
propose that the mutator synergy between exo1 and
pol3-01 is due to these exonucleases being able to act on
the same substrate. Therefore, an essential step in MMR (such as
removal of the mismatch) could not occur if both exonuclease functions
were inactivated. This view is supported by the observation that the
rate of reversion to Lys+ and His+ in the
pol3-01/pol3-01 exo1/exo1 double mutant is comparable to the
rate in the pol3-01/pol3-01 msh2/msh2 double mutant (Table 3).
Presented in Fig. 1 is a model describing
how Exo1 along with DNA Pol and/or DNA Pol could act to reduce
frameshift mutations in homonucleotide runs. The DNA Pol and Pol
3'5' proofreading exonuclease reduces the incidence of frameshift
intermediates at the time of replication. Frameshift intermediates that
escape proofreading are subsequently corrected by MMR. The synergy
between the pol2-4 (or pol3-01) and the
exo1 mutation in short homonucleotide runs (or in the
his7-2 reversion assay) can be explained by the participation of Exo1 in the removal of mismatches generated during replication by the proofreading-deficient Pol- or Pol-exonuclease (i.e., acting in series with proofreading). Although the proofreading exonucleases of Pol and Pol have no function in long
homonucleotide runs, we propose that they function along with the
5'3' exonuclease of Exo1 to excise mismatches in the MMR pathway.
The degradation process would be facilitated by DNA helicases, since
Exo1, Pol, and Pol nucleases degrade single-stranded DNA
efficiently (9, 22, 32). (In an E. coli
reconstituted MMR system, both a DNA helicase and exonucleases are
required for mismatch removal [5, 18].) In the
framework of this model, the stronger synergy of the pol2-4
or the pol3-01 mutation with the msh2 defect in
MMR for mutations in short as compared to long homonucleotide runs can
be due to pol2-4 or pol3-01 affecting both
proofreading and MMR in short homonucleotide runs, but only MMR in the
longer runs.
|
3'5' exonuclease with
Exo1 in error prevention is apparent for the long homonucleotide run
and provides strong support for our model, in which these nucleases are
involved in a late step of strand-specific mismatch removal. For the
long homonucleotide runs A12 and A14, in which Pol-exonuclease proofreading is inefficient, a defect in Exo1, Pol-exonuclease, or Pol-exonuclease alone results in a mutation rate that is <1% of that for an msh2 strain. The
pol2-4 exo1 double mutant exhibits mutation rates that are
10% of those seen with the pol2-4 msh2 strains (Table 1).
However the combination of pol3-01 with exo1 in a
diploid strain (the haploid strain is inviable) has an even more
profound effect, resulting in mutation rates that are comparable to the
most mutation-prone genetic combination, pol3-01 msh2
(mutation rates of the pol3-01 exo1 mutant are from 40 to
80% of those seen in the pol3-01 msh2 mutant; Table 3). Assuming that their impact on the mutation rate is directly due to a
role in MMR, our genetic results suggest that Pol-exonuclease and
Exo1 may be responsible for much of the mismatch excision during MMR.
The synergistic interaction between Pol-exonuclease and Exo1 in MMR
is similar to that found for Msh3 and Msh6. While the single mutant
msh3 and msh6 strains have a small effect on the
mutation rate, the msh3 msh6 double mutant exhibits a
mutator phenotype comparable to that of the msh2 mutant
(Table 1). We therefore suggest that Exo1 and Pol-exonucleases are
major exonucleases involved in the mismatch excision step.
Formally, it is possible that there are two separate pathways
(polymerase exonucleases plus Exo1 versus Msh2) of error avoidance, so
that defects in both pathways might lead to additive increases in
mutation rates. Because of the observed high error rates, it would be
difficult to distinguish epistasis (same pathway) from additivity
(separate pathways). Consistent with a single pathway is the fact that
the Lys+ frameshift reversion spectra in the
msh2, msh2 pol2-4, and exo1 pol2-4
strains are similar in that they exhibit hotspots in the A4
and A5 homonucleotide runs of the
lys2::insE-A4 and
lys2::insE-A5 alleles, respectively
(Table 1).
Roles of 5'3' and 3'5' nucleases in DNA metabolism.
We
and others have found that several combinations of nuclease mutations
can lead to lethality for haploid yeast cells (Table 4). The inviability of a double mutant
has been attributed to a high mutation rate resulting in error
catastrophe (8, 23, 24). In E. coli the
dnaQ926 mutation appears to cause lethality as a result of
the loss of proofreading and the subsequent saturation of DNA MMR.
Consistent with this, dnaQ926 strains are viable if they
carry a dnaE antimutator allele or a multicopy plasmid with the E. coli mutL gene (8).
|
-exonuclease and DNA
Pol-exonuclease participate in the removal of mismatches during MMR
and that DNA Pol is associated with MMR (20). It is
interesting that both DNA Pol and DNA Pol interact with PCNA
(6), which is also involved in MMR (12, 43). DNA
Pol and DNA Pol might be recruited to the MMR complex by an
interaction with PCNA or with other MMR proteins. Thus, mutations in
the putative MMR protein interaction domains of DNA Pol and DNA
Pol could lead to a defect in the excision step of MMR. Such
mutations might exhibit phenotypes similar to pol2-4 and
pol3-01 in combination with exo1 (i.e., instability of long homonucleotide runs but normal proofreading). Recently we identified such a mutation in the POL2 gene
located between the polymerase and checkpoint domains (14a).
A search for similar mutations in the POL3 gene is in progress.
Previously, it was reported that the pol2-4 pol3-01 haploid
double mutant was also inviable, but diploid double mutants are viable
and exhibit a mutator synergy (24). It was proposed that even if MMR is functional, a complete deficiency in proofreading of
both polymerases can lead to high mutation rates, resulting in error
catastrophe. As an alternative, we propose that the double defect in
pol2-4 pol3-01 leads not only to a loss of proofreading but
also to a defect in MMR; this reflects the participation of polymerase-associated exonucleases in MMR as described above (Fig. 1).
As summarized in Table 4, this and other studies demonstrate that while
defects in the individual Exo1, DNA Pol, and DNA Pol nucleases
may have a small effect, the combination of mutations can have a
profound impact on genome stability. Single mutations in these
exonucleases have only a moderate mutator phenotype in comparison with
a mutation in MSH2. We have shown that double mutations in
each pair of these three nuclease genes can lead to strong, synergistic
mutator effects or lethality for haploid yeast. The deletion of another
5'3' exonuclease gene, RAD27, in combination with
pol3-01 or exo1 mutations results in lethality (15, 38). In recent studies with this flap endonuclease, we have shown that it also exhibits synergy with either pol2-4
or msh2, suggesting a possible role in mismatch excision
(unpublished data).
Synergistic interactions affecting genome instability, such as those
described in this work, have important implications for the human
genome, in which genetic polymorphisms are common. Given the close
relationship between yeast and human genes involved in replication and
repair, particularly MMR, similar relationships may apply to the
equivalent pathways in human cells. For example, synergistic effects
might be expected for partially defective gene products that alone
cause a weak phenotype. These alleles would then cause a much more
serious deficiency and phenotype when combined with other weakly
defective proteins that act in overlapping genetic pathways.
| |
ACKNOWLEDGMENTS |
|---|
We thank M. Snyder for providing the disruption library, Y. Chernoff for the YEpHO plasmid, and J. Westmoreland and O. Kozyreva for help with experiments. We thank S. Bennett, J. Drake, R. Schaaper, and M. Sanders for helpful comments on the manuscript.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Chromosome Stability Group, Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences (NIEHS), 101 TW Alexander Dr., P.O. Box 12233, Research Triangle Park, NC 27709. Phone: (919) 541-4480. Fax: (919) 541-7593. E-mail: resnick{at}niehs.nih.gov.
Present address: Life Sensors Inc., Malvern, PA 19355.
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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