Hiep T. Tran

Hypermutability of homonucleotide runs in mismatch repair and DNA polymerase proofreading yeast mutants.

Homonucleotide runs in coding sequences are hot spots for frameshift mutations and potential sources of genetic changes leading to cancer in humans having a mismatch repair defect. We examined frameshift mutations in homonucleotide runs of deoxyadenosines ranging from 4 to 14 bases at the same position in the LYS2 gene of the yeast Saccharomyces cerevisiae. In the msh2 mismatch repair mutant, runs of 9 to 14 deoxyadenosines are 1,700-fold to 51,000-fold, respectively, more mutable for single-nucleotide deletions than are runs of 4 deoxyadenosines. These frameshift mutations can account for up to 99% of all forward mutations inactivating the 4-kb LYS2 gene. Based on results with single and double mutations of the POL2 and MSH2 genes, both DNA polymerase epsilon proofreading and mismatch repair are efficient for short runs while only the mismatch repair system prevents frameshift mutations in runs of > or = 8 nucleotides. Therefore, coding sequences containing long homonucleotide runs are likely to be at risk for mutational inactivation in cells lacking mismatch repair capability.

The 3′→5′ Exonucleases of DNA Polymerases δ and ɛ and the 5′→3′ Exonuclease Exo1 Have Major Roles in Postreplication Mutation Avoidance in Saccharomyces cerevisiae

ABSTRACT Replication fidelity is controlled by DNA polymerase proofreading and postreplication mismatch repair. We have genetically characterized the roles of the 5′→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 ɛ (pol2-4) and δ (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.

Destabilization of Yeast Micro- and Minisatellite DNA Sequences by Mutations Affecting a Nuclease Involved in Okazaki Fragment Processing (rad27) and DNA Polymerase δ (pol3-t)

ABSTRACT We examined the effects of mutations in the Saccharomyces cerevisiae RAD27 (encoding a nuclease involved in the processing of Okazaki fragments) and POL3 (encoding DNA polymerase δ) genes on the stability of a minisatellite sequence (20-bp repeats) and microsatellites (1- to 8-bp repeat units). Both therad27 and pol3-t mutations destabilized both classes of repeats, although the types of tract alterations observed in the two mutant strains were different. The tract alterations observed in rad27 strains were primarily additions, and those observed in pol3-t strains were primarily deletions. Measurements of the rates of repetitive tract alterations in strains with both rad27 and pol3-t indicated that the stimulation of microsatellite instability by rad27 was reduced by the effects of the pol3-t mutation. We also found that rad27 and pol3-01 (an allele carrying a mutation in the “proofreading” exonuclease domain of DNA polymerase δ) mutations were synthetically lethal.

Replication slippage between distant short repeats in Saccharomyces cerevisiae depends on the direction of replication and the RAD50 and RAD52 genes.

Small direct repeats, which are frequent in all genomes, are a potential source of genome instability. To study the occurrence and genetic control of repeat-associated deletions, we developed a system in the yeast Saccharomyces cerevisiae that was based on small direct repeats separated by either random sequences or inverted repeats. Deletions were examined in the LYS2 gene, using a set of 31- to 156-bp inserts that included inserts with no apparent potential for secondary structure as well as two quasipalindromes. All inserts were flanked by 6- to 9-bp direct repeats of LYS2 sequence, providing an opportunity for Lys+ reversion via precise excision. Reversions could arise by extended deletions involving either direct repeats or random sequences and by -1-or +2-bp frameshift mutations. The deletion breakpoints were always associated with short (3- to 9-bp) perfect or imperfect direct repeats. Compared with the POL+ strain, deletions between small direct repeats were increased as much as 100-fold, and the spectrum was changed in a temperature-sensitive DNA polymerase delta pol3-t mutant, suggesting a role for replication. The type of deletion depended on orientation relative to the origin of replication. On the basis of these results, we propose (i) that extended deletions between small repeats arise by replication slippage and (ii) that the deletions occur primarily in either the leading or lagging strand. The RAD50 and RAD52 genes, which are required for the recombinational repair of many kinds of DNA double-strand breaks, appeared to be required also for the production of up to 90% of the deletions arising between separated repeats in the pol3-t mutant, suggesting a newly identified role for these genes in genome stability and possibly replication.

A Novel Role in DNA Metabolism for the Binding of Fen1/Rad27 to PCNA and Implications for Genetic Risk

ABSTRACT Fen1/Rad27 nuclease activity, which is important in DNA metabolism, is stimulated by proliferating cell nuclear antigen (PCNA) in vitro. The in vivo role of the PCNA interaction was investigated in the yeast Rad27. A nuclease-defective rad27 mutation had a dominant-negative effect that was suppressed by a mutation in the PCNA binding site, thereby demonstrating the importance of the Rad27-PCNA interaction. The PCNA-binding defect alone had little effect on mutation, recombination, and the methyl methanesulfonate (MMS) response in repair-competent cells, but it greatly amplified the MMS sensitivity of a rad51 mutant. Furthermore, the PCNA binding mutation resulted in lethality when combined with a homozygous or even a heterozygous pol3-01 mutation in the 3′→5′ exonuclease domain of DNA polymerase δ. These results suggest that phenotypically mild polymorphisms in DNA metabolic proteins can have dramatic consequences when combined.

Altered replication and inverted repeats induce mismatch repair-independent recombination between highly diverged DNAs in yeast.

Replication, DNA organization, and mismatch repair (MMR) can influence recombination. We examined the effects of altered replication due to a mutation in the polymerase delta gene, long inverted repeats (LIRs) in motifs similar to those in higher eukaryotes, and MMR on intrachromosomal recombination between highly diverged (28%) truncated genes in Saccharomyces cerevisiae. A combination of altered replication and an LIR increased recombination up to 700-fold, while each alone led to a 3- to 20-fold increase. Homeologous recombination was not altered by pms1, msh2, and msh3 mismatch repair mutations. Similar to our previous observations for replication slippage-mediated deletions, there were > or = 5-bp identical runs at the recombination breakpoints. We propose that the dramatic increase in recombination results from enhancement of the effects of altered replication by the LIR, leading to recombinationally active initiating structures. Such interactions predict replication-related, MMR-independent genome changes.