SUMMARY FANCM remodels branched DNA structures and plays essential roles in the cellular response to DNA replication stress. Here we show that FANCM forms a conserved DNA remodeling complex with a histone-fold heterodimer, MHF. We find that MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. In vertebrates, FANCM-MHF associates with the Fanconi anemia (FA) core complex, promotes FANCD2 monoubiquitination in response to DNA damage, and suppresses sister-chromatid exchanges. Yeast orthologs of these proteins function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA remodeling complex that protects replication forks from yeast to human.

Accurate DNA synthesis by the replicative DNA polymerases α, δ, and ε is critical for genome stability in eukaryotes. In humans, over 20 SNPs were reported that result in amino-acid changes in Polδ or Polε. In addition, Polδ variants were found in colon-cancer cell lines and in sporadic colorectal carcinomas. Using the yeast-model system, we examined the functional consequences of two cancerassociated Polδ mutations and four polymorphisms affecting wellconserved regions of Polδ or Polε. We show that the R696W substitution in Polδ (analog of the R689W change in the human cancer-cell line DLD-1) is lethal in haploid and homozygous diploid yeast. The cell death results from a catastrophic increase in spontaneous mutagenesis attributed to low-fidelity DNA synthesis by Polδ-R696W. Heterozygotes survive, and the mutation rate depends on the relative expression level of wild-type versus mutant alleles. Based on these observations, we propose that the mutation rate in heterozygous human cells could be regulated by transient changes in gene expression leading to a temporary excess of Polδ-R689W. The similarities between the mutational spectra of the yeast strains producing Polδ-R696W and DLD-1 cells suggest that the altered Polδ could be responsible for a significant proportion of spontaneous mutations in this cancer cell line. These results suggest that the highly error-prone Polδ-R689W could contribute to cancer initiation and/or progression in humans.

Trinucleotide repeats (TNRs) are unique DNA microsatellites that can expand to cause human disease. Recently, Srs2 was identified as a protein that inhibits TNR expansions in Saccharomyces cerevisiae. Here, we demonstrate that Srs2 inhibits CAG · CTG expansions in conjunction with the error-free branch of postreplication repair (PRR). Like srs2 mutants, expansions are elevated in rad18 and rad5 mutants, as well as the PRR-specific PCNA alleles pol30-K164R and pol30-K127/164R. Epistasis analysis indicates that Srs2 acts upstream of these PRR proteins. Also, like srs2 mutants, the pol30-K127/164R phenotype is specific for expansions, as this allele does not alter mutation rates at dinucleotide repeats, at nonrepeating sequences, or for CAG · CTG repeat contractions. Our results suggest that Srs2 action and PRR processing inhibit TNR expansions. We also investigated the relationship between PRR and Rad27 (Fen1), a well-established inhibitor of TNR expansions that acts at 5 flaps. Our results indicate that PRR protects against expansions arising from the 3 terminus, presumably replication slippage events. This work provides the first evidence that CAG · CTG expansions can occur by 3 slippage, and our results help define PRR as a key cellular mechanism that protects against expansions.Expansions of trinucleotide repeats (TNRs) are the genetic basis for at least 15 human neurological diseases, including Huntington's disease, myotonic dystrophy type 1, and fragile X syndrome (39,42,60). Inheritance patterns in families afflicted with these diseases show an ongoing mutational process where the disease-causing allele tends to increase in length in each generation. This dynamic mutation pattern (47, 48) results in the non-Mendelian inheritance behavior called anticipation, which is a signature of this class of diseases. TNR tracts have several unique mutational properties in affected families. First, TNR mutations are locus specific. For example, the HD locus is highly unstable in Huntington's patients, but mutations are rare elsewhere in the genome, including other DNA microsatellites (16). DNA sequence is also critical. Fourteen of the diseases in this class are caused by mutation of the repeat sequence CNG (39,42,60), where N corresponds to any nucleotide. The sole exception, Friedreich's ataxia, has an unstable GAA sequence (8). This sequence restriction correlates strongly with the ability of these sequences to form hairpins and other unusual secondary structures in vitro (15). Accordingly, these secondary structures are thought to be a critical component of the expansion mechanism (39, 42, 60). Together, locus specificity and sequence restriction indicate that DNA metabolism at TNR sequences is unusual, compared to the rest of the genome. Thus, new rules appear to govern how cellular proteins process TNRs to either promote or prevent expansions.Since TNR expansions require the addition of DNA, clearly the mechanism must involve a DNA synthetic event. Various models of TNR instability invoke replication and/or repair...

Background: Yeast proteins homologous to human Fanconi proteins exist, but their cross-link repair functions are undefined. Results: Mutants are cross-link-sensitive, and Mph1 overexpression protects yeast cells. Conclusion:The yeast pathway is epistatic with rad5 and rad51, and the Mph1 helicase stabilizes ICL-stalled replication forks in a Rad5-dependent manner. Significance: Rad5 directs the yeast Fanconi-like interstrand cross-link repair pathway.