Saccharomyces cerevisiae pol30 (Proliferating Cell Nuclear Antigen) Mutations Impair Replication Fidelity and Mismatch Repair

To understand the role of POL30 in mutation suppression, 11 Saccharomyces cerevisiae pol30 mutator mutants were characterized. These mutants were grouped based on their mutagenic defects. Many pol30 mutants harbor multiple mutagenic defects and were placed in more than one group. Group A mutations (pol30-52, -104, -108, and -126) caused defects in mismatch repair (MMR). These mutants exhibited mutation rates and spectra reminiscent of MMR-defective mutants and were defective in an in vivo MMR assay. The mutation rates of group A mutants were enhanced by a msh2 or a msh6 mutation, indicating that MMR deficiency is not the only mutagenic defect present. Group B mutants (pol30-45, -103, -105, -126, and -114) exhibited increased accumulation of either deletions alone or a combination of deletions and duplications (4 to 60 bp). All deletion and duplication breakpoints were flanked by 3 to 7 bp of imperfect direct repeats. Genetic analysis of one representative group B mutant, pol30-126, suggested polymerase slippage as the likely mutagenic mechanism. Group C mutants (pol30-100, -103, -105, -108, and -114) accumulated base substitutions and exhibited synergistic increases in mutation rate when combined with msh6 mutations, suggesting increased DNA polymerase misincorporation as a mutagenic defect. The synthetic lethality between a group A mutant, pol30-104, and rad52 was almost completely suppressed by the inactivation of MSH2. Moreover, pol30-104 caused a hyperrecombination phenotype that was partially suppressed by a msh2 mutation. These results suggest that pol30-104 strains accumulate DNA breaks in a MSH2-dependent manner.     

Msh2 separation of function mutations confer defects in the initiation steps of mismatch repair

In eukaryotes the MSH2-MSH3 and MSH2-MSH6 heterodimers initiate mismatch repair (MMR) by recognizing and binding to DNA mismatches. The MLH1-PMS1 heterodimer then interacts with the MSH proteins at or near the mismatch site and is thought to act as a mediator to recruit downstream repair proteins. Here we analyzed five msh2 mutants that are functional in removing 3' non-homologous tails during double-strand break repair but are completely defective in MMR. Because non-homologous tail removal does not require MSH6, MLH1, or PMS1 functions, a characterization of the msh2 separation of function alleles should provide insights into early steps in MMR. Using the Taq MutS crystal structure as a model, three of the msh2 mutations, msh2-S561P, msh2-K564E, msh2-G566D, were found to map to a domain in MutS involved in stabilizing mismatch binding. Gel mobility shift and DNase I footprinting assays showed that two of these mutations conferred strong defects on MSH2-MSH6 mismatch binding. The other two mutations, msh2-S656P and msh2-R730W, mapped to the ATPase domain. DNase I footprinting, ATP hydrolysis, ATP binding, and MLH1-PMS1 interaction assays indicated that the msh2-S656P mutation caused defects in ATP-dependent dissociation of MSH2-MSH6 from mismatch DNA and in interactions between MSH2-MSH6 and MLH1-PMS1. In contrast, the msh2-R730W mutation disrupted MSH2-MSH6 ATPase activity but did not strongly affect ATP binding or interactions with MLH1-PMS1. These results support a model in which MMR can be dissected into discrete steps: stable mismatch binding and sensing, MLH1-PMS1 recruitment, and recycling of MMR components.     

exo1-Dependent Mutator Mutations: Model System for Studying Functional Interactions in Mismatch Repair

EXO1 interacts with MSH2 and MLH1 and has been proposed to be a redundant exonuclease that functions in mismatch repair (MMR). To better understand the role of EXO1 in mismatch repair, a genetic screen was performed to identify mutations that increase the mutation rates caused by weak mutator mutations such as exo1Delta and pms1-A130V mutations. In a screen starting with an exo1 mutation, exo1-dependent mutator mutations were obtained in MLH1, PMS1, MSH2, MSH3, POL30 (PCNA), POL32, and RNR1, whereas starting with the weak pms1 allele pms1-A130V, pms1-dependent mutator mutations were identified in MLH1, MSH2, MSH3, MSH6, and EXO1. These mutations only cause weak MMR defects as single mutants but cause strong MMR defects when combined with each other. Most of the mutations obtained caused amino acid substitutions in MLH1 or PMS1, and these clustered in either the ATP-binding region or the MLH1-PMS1 interaction regions of these proteins. The mutations showed two other types of interactions: specific pairs of mutations showed unlinked noncomplementation in diploid strains, and the defect caused by pairs of mutations could be suppressed by high-copy-number expression of a third gene, an effect that showed allele and overexpressed gene specificity. These results support a model in which EXO1 plays a structural role in MMR and stabilizes multiprotein complexes containing a number of MMR proteins. A similar role is proposed for PCNA based on the data presented.     

Saccharomyces cerevisiae MSH2-MSH3 and MSH2-MSH6 complexes display distinct requirements for DNA binding domain I in mismatch recognition

In eukaryotic mismatch repair (MMR) MSH2-MSH6 initiates the repair of base-base and small insertion/deletion mismatches while MSH2-MSH3 repairs larger insertion/deletion mismatches. Here, we show that the msh2Delta1 mutation, containing a complete deletion of the conserved mismatch recognition domain I of MSH2, conferred a separation of function phenotype with respect to MSH2-MSH3 and MSH2-MSH6 functions. Strains bearing the msh2Delta1 mutation were nearly wild-type in MSH2-MSH6-mediated MMR and in suppressing recombination between DNA sequences predicted to form mismatches recognized by MSH2-MSH6. However, these strains were completely defective in MSH2-MSH3-mediated MMR and recombination functions. This information encouraged us to analyze the contributions of domain I to the mismatch binding specificity of MSH2-MSH3 in genetic and biochemical assays. We found that domain I in MSH2 contributed a non-specific DNA binding activity while domain I of MSH3 appeared important for mismatch binding specificity and for suppressing non-specific DNA binding. These observations reveal distinct requirements for the MSH2 DNA binding domain I in the repair of DNA mismatches and suggest that the binding of MSH2-MSH3 to mismatch DNA involves protein-DNA contacts that appear very different from those required for MSH2-MSH6 mismatch binding.     

A mutational analysis of the yeast proliferating cell nuclear antigen indicates distinct roles in DNA replication and DNA repair.

The saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA), encoded by the POL30 gene, is essential for DNA replication and DNA repair processes. Twenty-one site-directed mutations were constructed in the POL30 gene, each mutation changing two adjacently located charged amino acids to alanines. Although none of the mutant strains containing these double-alanine mutations as the sole source of PCNA were temperature sensitive or cold sensitive for growth, about a third of the mutants showed sensitivity to UV light. Some of those UV-sensitive mutants had elevated spontaneous mutation rates. In addition, several mutants suppressed a cold-sensitive mutation in the CDC44 gene, which encodes the large subunit of replication factor C. A cold-sensitive mutant, which was isolated by random mutagenesis, showed a terminal phenotype at the restrictive temperature consistent with a defect in DNA replication. Several mutant PCNAs were expressed and purified from Escherichia coli, and their in vitro properties were determined. The cold-sensitive mutant (pol30-52, S115P) was a monomer, rather than a trimer, in solution. This mutant was deficient for DNA synthesis in vitro. Partial restoration of DNA polymerase delta holoenzyme activity was achieved at 37 degrees C but not at 14 degrees C by inclusion of the macromolecular crowding agent polyethylene glycol in the assay. The only other mutant (pol30-6, DD41,42AA) that showed a growth defect was partially defective for interaction with replication factor C and DNA polymerase delta but completely defective for interaction with DNA polymerase epsilon. Two other mutants sensitive to DNA damage showed no defect in vitro. These results indicate that the latter mutants are specifically impaired in one or more DNA repair processes whereas pol30-6 and pol30-52 mutants show their primary defects in the basic DNA replication machinery with probable associated defects in DNA repair. Therefore, DNA repair requires interactions between repair-specific protein(s) and PCNA, which are distinct from those required for DNA replication.     

Multiple factors insulate Msh2-Msh6 mismatch repair activity from defects in Msh2 domain I

DNA mismatch repair (MMR) is a highly conserved mutation avoidance mechanism that corrects DNA polymerase misincorporation errors. In initial steps in MMR, Msh2-Msh6 binds mispairs and small insertion/deletion loops, and Msh2-Msh3 binds larger insertion/deletion loops. The msh2Δ1 mutation, which deletes the conserved DNA-binding domain I of Msh2, does not dramatically affect Msh2-Msh6-dependent repair. In contrast, msh2Δ1 mutants show strong defects in Msh2-Msh3 functions. Interestingly, several mutations identified in patients with hereditary non-polyposis colorectal cancer map to domain I of Msh2; none have been found in MSH3. To understand the role of Msh2 domain I in MMR, we examined the consequences of combining the msh2Δ1 mutation with mutations in two distinct regions of MSH6 and those that increase cellular mutational load (pol3-01 and rad27). These experiments reveal msh2Δ1-specific phenotypes in Msh2-Msh6 repair, with significant effects on mutation rates. In vitro assays demonstrate that msh2Δ1-Msh6 DNA binding is less specific for DNA mismatches and produces an altered footprint on a mismatch DNA substrate. Together, these results provide evidence that, in vivo, multiple factors insulate MMR from defects in domain I of Msh2 and provide insights into how mutations in Msh2 domain I may cause hereditary non-polyposis colorectal cancer.     

A mutation in the MSH6 subunit of the Saccharomyces cerevisiae MSH2-MSH6 complex disrupts mismatch recognition.

In yeast, MSH2 interacts with MSH6 to repair base pair mismatches and single nucleotide insertion/deletion mismatches and with MSH3 to recognize small loop insertion/deletion mismatches. We identified a msh6 mutation (msh6-F337A) that when overexpressed in wild type strains conferred a defect in both MSH2-MSH6- and MSH2-MSH3-dependent mismatch repair pathways. Genetic analysis suggested that this phenotype was due to msh6-F337A sequestering MSH2 and preventing it from interacting with MSH3 and MSH6. In UV cross-linking, filter binding, and gel retardation assays, the MSH2-msh6-F337A complex displayed a mismatch recognition defect. These observations, in conjunction with ATPase and dissociation rate analysis, suggested that MSH2-msh6-F337A formed an unproductive complex that was unable to stably bind to mismatch DNA.     

Role of proliferating cell nuclear antigen interactions in the mismatch repair-dependent processing of mitotic and meiotic recombination intermediates in yeast

The mismatch repair (MMR) system is critical not only for the repair of DNA replication errors, but also for the regulation of mitotic and meiotic recombination processes. In a manner analogous to its ability to remove replication errors, the MMR system can remove mismatches in heteroduplex recombination intermediates to generate gene conversion events. Alternatively, such mismatches can trigger an MMR-dependent antirecombination activity that blocks the completion of recombination, thereby limiting interactions between diverged sequences. In Saccharomyces cerevisiae, the MMR proteins Msh3, Msh6, and Mlh1 interact with proliferating cell nuclear antigen (PCNA), and mutations that disrupt these interactions result in a mutator phenotype. In addition, some mutations in the PCNA-encoding POL30 gene increase mutation rates in an MMR-dependent manner. In the current study, pol30, mlh1, and msh6 mutants were used to examine whether MMR-PCNA interactions are similarly important during mitotic and meiotic recombination. We find that MMR-PCNA interactions are important for repairing mismatches formed during meiotic recombination, but play only a relatively minor role in regulating the fidelity of mitotic recombination.     

The Prevention of Repeat-Associated Deletions in Saccharomyces Cerevisiae by Mismatch Repair Depends on Size and Origin of Deletions

We have investigated the effects of mismatch repair on 1- to 61-bp deletions in the yeast Saccharomyces cerevisiae. The deletions are likely to involve unpaired loop intermediates resulting from DNA polymerase slippage. The mutator effects of mutations in the DNA polymerase δ (POL3) gene and the recombinational repair RAD52 gene were studied in combination with mismatch repair defects. The pol3-t mutation increased up to 1000-fold the rate of extended (7-61 bp) but not of 1-bp deletions. In a rad52 null mutant only the 1-bp deletions were increased (12-fold). The mismatch repair mutations pms1, msh2 and msh3 did not affect 31- and 61-bp deletions in the pol3-t but increased the rates of 7- and 1-bp deletions. We propose that loops less than or equal to seven bases generated during replication are subject to mismatch repair by the PMS1, MSH2, MSH3 system and that it cannot act on loops >=31 bases. In contrast to the pol3-t, the enhancement of 1-bp deletions in a rad52 mutant is not altered by a pms1 mutation. Thus, mismatch repair appears to be specific to errors of DNA synthesis generated during semiconservative replication.     

EXO1 and MSH6 are high-copy suppressors of conditional mutations in the MSH2 mismatch repair gene of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, Msh2p, a central component in mismatch repair, forms a heterodimer with Msh3p to repair small insertion/deletion mismatches and with Msh6p to repair base pair mismatches and single-nucleotide insertion/deletion mismatches. In haploids, a msh2Delta mutation is synthetically lethal with pol3-01, a mutation in the Poldelta proofreading exonuclease. Six conditional alleles of msh2 were identified as those that conferred viability in pol3-01 strains at 26 degrees but not at 35 degrees. DNA sequencing revealed that mutations in several of the msh2(ts) alleles are located in regions with previously unidentified functions. The conditional inviability of two mutants, msh2-L560S pol3-01 and msh2-L910P pol3-01, was suppressed by overexpression of EXO1 and MSH6, respectively. Partial suppression was also observed for the temperature-sensitive mutator phenotype exhibited by msh2-L560S and msh2-L910P strains in the lys2-Bgl reversion assay. High-copy plasmids bearing mutations in the conserved EXO1 nuclease domain were unable to suppress msh2-L560S pol3-01 conditional lethality. These results, in combination with a genetic analysis of msh6Delta pol3-01 and msh3Delta pol3-01 strains, suggest that the activity of the Msh2p-Msh6p heterodimer is important for viability in the presence of the pol3-01 mutation and that Exo1p plays a catalytic role in Msh2p-mediated mismatch repair.     

Mutations in yeast proliferating cell nuclear antigen define distinct sites for interaction with DNA polymerase delta and DNA polymerase epsilon.

The importance of the interdomain connector loop and of the carboxy-terminal domain of Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) for functional interaction with DNA polymerases delta (Poldelta) and epsilon (Pol epsilon) was investigated by site-directed mutagenesis. Two alleles, pol30-79 (IL126,128AA) in the interdomain connector loop and pol30-90 (PK252,253AA) near the carboxy terminus, caused growth defects and elevated sensitivity to DNA-damaging agents. These two mutants also had elevated rates of spontaneous mutations. The mutator phenotype of pol30-90 was due to partially defective mismatch repair in the mutant. In vitro, the mutant PCNAs showed defects in DNA synthesis. Interestingly, the pol30-79 mutant PCNA (pcna-79) was most defective in replication with Poldelta, whereas pcna-90 was defective in replication with Pol epsilon. Protein-protein interaction studies showed that pcna-79 and pcna-90 failed to interact with Pol delta and Pol epsilon, respectively. In addition, pcna-90 was defective in interaction with the FEN-1 endo-exonuclease (RTH1 product). A loss of interaction between pcna-79 and the smallest subunit of Poldelta, the POL32 gene product, implicates this interaction in the observed defect with the polymerase. Neither PCNA mutant showed a defect in the interaction with replication factor C or in loading by this complex. Processivity of DNA synthesis by the mutant holoenzyme containing pcna-79 was unaffected on poly(dA) x oligo(dT) but was dramatically reduced on a natural template with secondary structure. A stem-loop structure with a 20-bp stem formed a virtually complete block for the holoenzyme containing pcna-79 but posed only a minor pause site for wild-type holoenzyme, indicating a function of the POL32 gene product in allowing replication past structural blocks.     

The RAD52 recombinational repair pathway is essential in pol30 (PCNA) mutants that accumulate small single-stranded DNA fragments during DNA synthesis.

To identify in vivo pathways that compensate for impaired proliferating cell nuclear antigen (PCNA or Pol30p in yeast) activity, we performed a synthetic lethal screen with the yeast pol30-104 mutation. We identified nine mutations that display synthetic lethality with pol30-104; three mutations affected the structural gene for the large subunit of replication factor C (rfc1), which loads PCNA onto DNA, and six mutations affected three members of the RAD52 epistasis group for DNA recombinational repair (rad50, rad52 and rad57). We also found that pol30-104 displayed synthetic lethality with mutations in other members of the RAD52 epistasis group (rad51 and rad54), but not with mutations in members of the RAD3 nor the RAD6 epistasis group. Analysis of nine different pol30 mutations shows that the requirement for the RAD52 pathway is correlated with a DNA replication defect but not with the relative DNA repair defect caused by pol30 mutations. In addition, mutants that require RAD52 for viability (pol30-100, pol30-104, rfc1-1 and rth1delta) accumulate small single-stranded DNA fragments during DNA replication in vivo. Taken together, these data suggest that the RAD52 pathway is required when there are defects in the maturation of Okazaki fragments.     

Spontaneous frameshift mutations in Saccharomyces cerevisiae: accumulation during DNA replication and removal by proofreading and mismatch repair activities

The msh6 mismatch repair gene of Schizosaccharomyces pombe was cloned, sequenced, and inactivated. Strains bearing all combinations of inactivated msh6, msh2, and swi4 (the S. pombe MSH3 ortholog) alleles were tested for their defects in mitotic and meiotic mismatch repair. Mitotic mutation rates were similarly increased in msh6 and msh2 mutants, both for reversion of a base-base substitution as well as of an insertion of one nucleotide in a mononucleotide run. Tetrad analysis and intragenic two-factor crosses revealed that meiotic mismatch repair was affected in msh6 to the same extent as in msh2 background. In contrast, loss of Swi4 likely did not cause a defect in mismatch repair, but rather resulted in reduced recombination frequency. Consistently, a mutated swi4 caused a two- to threefold reduction of recombinants in intergenic crosses, while msh2 and msh6 mutants were not significantly different from wild type. In summary, our study showed that Msh6 plays the same important role as Msh2 in the major mismatch repair pathway of S. pombe, while Swi4 rather functions in recombination.     

Biochemical analysis of the human mismatch repair proteins hMutSα MSH2(G674A)-MSH6 and MSH2-MSH6(T1219D)

The heterodimeric human MSH2-MSH6 protein initiates DNA mismatch repair (MMR) by recognizing mismatched bases that result from replication errors. Msh2(G674A) or Msh6(T1217D) mice that have mutations in or near the ATP binding site of MSH2 or ATP hydrolysis catalytic site of MSH6 develop cancer and have a reduced lifespan due to loss of the MMR pathway (Lin, D. P., Wang, Y., Scherer, S. J., Clark, A. B., Yang, K., Avdievich, E., Jin, B., Werling, U., Parris, T., Kurihara, N., Umar, A., Kucherlapati, R., Lipkin, M., Kunkel, T. A., and Edelmann, W. (2004) Cancer Res. 64, 517-522; Yang, G., Scherer, S. J., Shell, S. S., Yang, K., Kim, M., Lipkin, M., Kucherlapati, R., Kolodner, R. D., and Edelmann, W. (2004) Cancer Cell 6, 139-150). Mouse embryonic fibroblasts from these mice retain an apoptotic response to DNA damage. Mutant human MutSα proteins MSH2(G674A)-MSH6(wt) and MSH2(wt)-MSH6(T1219D) are profiled in a variety of functional assays and as expected fail to support MMR in vitro, although they retain mismatch recognition activity. Kinetic analyses of DNA binding and ATPase activities and examination of the excision step of MMR reveal that the two mutants differ in their underlying molecular defects. MSH2(wt)-MSH6(T1219D) fails to couple nucleotide binding and mismatch recognition, whereas MSH2(G674A)-MSH6(wt) has a partial defect in nucleotide binding. Nevertheless, both mutant proteins remain bound to the mismatch and fail to promote efficient excision thereby inhibiting MMR in vitro in a dominant manner. Implications of these findings for MMR and DNA damage signaling by MMR proteins are discussed.     

Separation-of-Function Mutations in Saccharomyces cerevisiae MSH2 That Confer Mismatch Repair Defects but Do Not Affect Nonhomologous-Tail Removal during Recombination

Yeast Msh2p forms complexes with Msh3p and Msh6p to repair DNA mispairs that arise during DNA replication. In addition to their role in mismatch repair (MMR), the MSH2 and MSH3 gene products are required to remove 3' nonhomologous DNA tails during genetic recombination. The mismatch repair genes MSH6, MLH1, and PMS1, whose products interact with Msh2p, are not required in this process. We have identified mutations in MSH2 that do not disrupt genetic recombination but confer a strong defect in mismatch repair. Twenty-four msh2 mutations that conferred a dominant negative phenotype for mismatch repair were isolated. A subset of these mutations mapped to residues in Msh2p that were analogous to mutations identified in human nonpolyposis colorectal cancer msh2 kindreds. Approximately half of the these MMR-defective mutations retained wild-type or nearly wild-type activity for the removal of nonhomologous DNA tails during genetic recombination. The identification of mutations in MSH2 that disrupt mismatch repair without affecting recombination provides a first step in dissecting the Msh-effector protein complexes that are thought to play different roles during DNA repair and genetic recombination.     

Computational investigation on effect of mutations in PCNA resulting in structural perturbations and inhibition of mismatch repair pathway

Abstract

From bacteria to mammals, DNA mismatch repair (MMR) pathway plays an essential role in eliminating mismatched nucleotides and insertion-deletion mismatches during the process of DNA replication. Among many of the proteins which participate in the mismatch repair process, proliferating cell nuclear antigen (PCNA) remains the principal conductor at the replication fork. The pol30-201 and pol30-204 are the two mutated alleles which encode for C22Y and C81R mutant forms of PCNA proteins. We performed long term molecular dynamics (MD) simulations analysis (0.8 μs) to understand the dynamic behavior and alterations in the structure of wild type and mutated forms of PCNA at the atomic level. We observed changes in the structural characteristics like length, radius, rise per residue of alpha helices in both the mutated forms of PCNA. Apart from it, disfigurement of the charge distribution which effects binding with the dsDNA due to mutant C22Y and other structural perturbations were also seen in regions significant for the formation of a biologically active trimeric form of PCNA due to mutant C81R. Our analysis of native and mutated forms of PCNA provides an insight into the essential structural and functional features required for proper and well-coordinated DNA mismatch repair process and consequences of the mutation leading to an impaired process of MMR. These structural characteristics are fundamental for the MMR process and hence our analysis likely contributes to or presents the novel mechanism involved in the process of MMR.

Communicated by Ramaswamy H. Sarma     

In Vivo Analysis Reveals That the Interdomain Region of the Yeast Proliferating Cell Nuclear Antigen Is Important for DNA Replication and DNA Repair

To identify the regions of the proliferating cell nuclear antigen (PCNA) that are important for function in vivo, we used random mutagenesis to isolate 10 cold-sensitive (Cs(-)) and 31 methyl methanesulfonate-sensitive (Mms(s)) mutations of the PCNA gene (POL30) in Saccharomyces cerevisiae. Unlike the Mms(s) mutations, the Cs(-) mutations are strikingly clustered in the interdomain region of the three-dimensional PCNA monomer structure. At the restrictive temperature, the Cs(-) pol30 mutants undergo a RAD9-dependent arrest as large-budded cells with a 2c DNA content. Defects in DNA synthesis are suggested by a significant delay in the progression of synchronized pol30 cells through S phase at the restrictive temperature. DNA repair defects are revealed by the observation that Cs(-) pol30 mutants are very sensitive to the alkylating agent MMS and mildly sensitive to ultraviolet radiation, although they are not sensitive to gamma radiation. Finally, analysis of the chromosomal DNA in pol30 cells by velocity sedimentation gradients shows that pol30 cells accumulate single-stranded DNA breaks at the restrictive temperature. Thus, our results show that PCNA plays an essential role in both DNA replication and DNA repair in vivo.     

Genomic instability induced by mutations in Saccharomyces cerevisiae POL1

Mutations of chromosome replication genes can be one of the early events that promote genomic instability. Among genes that are involved in chromosomal replication, DNA polymerase alpha is essential for initiation of replication and lagging-strand synthesis. Here we examined the effect of two mutations in S. cerevisiae POL1, pol1-1 and pol1-17, on a microsatellite (GT)(16) tract. The pol1-17 mutation elevated the mutation rate 13-fold by altering sequences both inside and downstream of the (GT)(16) tract, whereas the pol1-1 mutation increased the mutation rate 54-fold by predominantly altering sequences downstream of the (GT)(16) tract in a RAD52-dependent manner. In a rad52 null mutant background pol1-1 and pol1-17 also exhibited different plasmid and chromosome loss phenotypes. Deletions of mismatch repair (MMR) genes induce a differential synergistic increase in the mutation rates of pol1-1 and pol1-17. These findings suggest that perturbations of DNA replication in these two pol1 mutants are caused by different mechanisms, resulting in various types of mutations. Thus, mutations of POL1 can induce a variety of mutator phenotypes and can be a source of genomic instability in cells.     

Proliferating cell nuclear antigen and ASF1 modulate silent chromatin in Saccharomyces cerevisiae via lysine 56 on histone H3

The formation and stability of epigenetically regulated chromatin is influenced by DNA replication and factors that modulate post-translational modifications on histones. Here we describe evidence that PCNA can affect silencing in Saccharomyces cerevisiae by facilitating deposition of H3 K56ac onto chromosomes. We propose that PCNA participates in this process through a pathway that includes replication factor C, the chromatin assembly factor Asf1p, and the K56-specific acetyltransferase Rtt109p. We show that mutation of POL30 or loss of K56-acetylation in rtt109 and histone H3 mutants enhances silencing at the crippled HMR locus HMRae via restoring Sir binding and that pol30 mutants with silencing phenotypes have reduced levels of H3 K56ac. Although loss of acetylation on H3 K56 was generally compatible with silencing, mutations at this residue also led to defects in silencing an ADE2 reporter at HMR and abolished silencing when combined with cac1 or pol30-8. These silencing phenotypes are analogous to those in asf1 mutants or pol30-6 and pol30-79 mutants with defects in ASF1-dependent pathways. On the basis of these findings, we propose that mutations in DNA replication factors alter acetylation of H3 K56. We show that this defect, in turn, contributes to misregulation of epigenetic processes as well as of cellular responses to DNA damage.