Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine if ribonucleotides are incorporated during DNA replication in vivo, we substituted leucine or glycine for an active site methionine in yeast DNA polymerase ε (Pol ε). Compared to wild type Pol ε, ribonucleotide incorporation in vitro was 3-fold lower for M644L and 11-fold higher for M644G Pol ε. This hierarchy was re-capitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201Δ strain progressed more slowly through S-phase, had elevated dNTP pools and generated 2–5 base pair deletions in repetitive sequences at a high rate and gene orientation-dependent manner. The data indicate that ribonucleotides are incorporated during replication in vivo, that they are removed by RNase H2-dependent repair, and that defective repair results in replicative stress and genome instability via DNA strand misalignment.

A two-hybrid system was used to screen yeast and human expression libraries for proteins that interact with mismatch repair proteins. PCNA was recovered from both libraries and shown in the case of yeast to interact with both MLH1 and MSH2. A yeast strain containing a mutation in the PCNA gene had a strongly elevated mutation rate in a dinucleotide repeat, and the rate was not further elevated in a strain also containing a mutation in MLH1. Mismatch repair activity was examined in human cell extracts using an assay that does not require DNA repair synthesis. Activity was inhibited by p21WAF1 or a p21 peptide, both of which bind to PCNA, and activity was restored to inhibited reactions by addition of PCNA. The data suggest a PCNA requirement in mismatch repair at a step preceding DNA resynthesis. The ability of PCNA to bind to MLH1 and MSH2 may reflect linkage between mismatch repair and replication and may be relevant to the roles of mismatch repair proteins in other DNA transactions.

Summary The Fanconi Anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA surrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 and EXDL2. FAN1 co-localizes at sites of DNA damage with the ID complex in a manner dependent on FAN1’s ubiquitin binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 5′-3′ exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain.

The RNase H class of enzymes degrades the RNA component of RNA:DNA hybrids and is important in nucleic acid metabolism. RNase H2 is specialized to remove single ribonucleotides (rNMPs) from duplex DNA, and its absence in budding yeast has been associated with the accumulation of deletions within short tandem repeats. Here, we demonstrate that rNMP-associated deletion formation requires the activity of Top1, a topoisomerase that relaxes supercoils by reversibly nicking duplex DNA. The reported studies extend the role of Top1 to include the processing of rNMPs in genomic DNA into irreversible single-strand breaks, an activity that can have distinct mutagenic consequences and may be relevant to human disease.

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Cyclin D1 is a component of the core cell cycle machinery1. Abnormally high levels of cyclin D1 are detected in many human cancer types2. To elucidate the molecular functions of cyclin D1 in human cancers, here we performed a proteomic screen for cyclin D1 protein partners in several types of human tumors. Analyses of cyclin D1-interactors revealed a network of DNA repair proteins, including RAD51, a recombinase that drives the homologous recombination process3. We found that cyclin D1 directly binds RAD51, and that cyclin D1-RAD51 interaction is induced by radiation. Like RAD51, cyclin D1 is recruited to DNA damage sites in a BRCA2-dependent fashion. Reduction of cyclin D1 levels in human cancer cells impaired recruitment of RAD51 to damaged DNA, impeded the homologous recombination-mediated DNA repair, and increased sensitivity of cells to radiation in vitro and in vivo. This effect was seen in cancer cells lacking the retinoblastoma protein, which do not require D-cyclins for proliferation4, 5. These findings reveal an unexpected function of a core cell cycle protein in DNA repair and suggest that targeting cyclin D1 may be beneficial also in retinoblastoma-negative cancers which are currently thought to be oblivious to cyclin D1 inhibition.

Exonuclease 1 (Exo1) is a 5-3 exonuclease that interacts with MutS and MutL homologs and has been implicated in the excision step of DNA mismatch repair. To investigate the role of Exo1 in mammalian mismatch repair and assess its importance for tumorigenesis and meiosis, we generated an Exo1 mutant mouse line. Analysis of Exo1 −/− cells for mismatch repair activity in vitro showed that Exo1 is required for the repair of base:base and single-base insertion/deletion mismatches in both 5 and 3 nick-directed repair. The repair defect in Exo1 −/− cells also caused elevated microsatellite instability at a mononucleotide repeat marker and a significant increase in mutation rate at the Hprt locus. Exo1 −/− animals displayed reduced survival and increased susceptibility to the development of lymphomas. In addition, Exo1 −/− male and female mice were sterile because of a meiotic defect. Meiosis in Exo1 −/− animals proceeded through prophase I; however, the chromosomes exhibited dynamic loss of chiasmata during metaphase I, resulting in meiotic failure and apoptosis. Our results show that mammalian Exo1 functions in mutation avoidance and is essential for male and female meiosis. The DNA mismatch repair (MMR) system is important for maintaining the integrity of the genome in prokaryotes and eukaryotes. It has evolved to correct mispaired bases that result from errors during DNA replication, DNA recombination, and from certain types of DNA damage. Analyses in yeast and mice also revealed an essential role of some eukaryotic MMR gene products in the control of meiotic recombination. The importance of MMR to mammals is also highlighted by the observation that germ-line mutations in several of the MMR genes are associated with hereditary nonpolyposis colorectal cancer (HNPCC), and the loss of MMR function underlies several types of sporadic cancers (Peltomaki and Vasen 1997;Peltomaki 2001).MMR was initially characterized in bacteria (Modrich 1991;Modrich and Lahue 1996) and more recently in eukaryotic cells (Kolodner 1996). The studies of mammalian MMR focused mainly on the role of the eukaryotic MutS and MutL homologs (MSH and MLH, respectively) in the initiation of the repair reaction. These analyses showed that the early steps of MMR include the recognition of mispaired nucleotide(s) by two heterodimeric complexes: MSH2-MSH6 functions in the repair of base:base mispairs as well as a range of insertion/deletion loop mispairs (IDLs), whereas MSH2-MSH3 primarily functions in the repair of IDLs (Marsischky et al. 1996;Genschel et al. 1998;Umar et al. 1998). Subsequent to mismatch recognition, these two MutS complexes interact with MutL complexes consisting of MLH1-PMS2 or MLH1-MLH3 (Prolla et al. 1994;Li and Modrich 1995;Flores-Rozas and Kolodner 1998;Wang et al. 1999). These interactions are absolutely necessary for the activation of downstream events including the excision of the misincorporated nucleotide(s) and filling in of the resulting single-strand gap by DNA resynthesis. These later steps in MMR as well as the nature...

Most errors that arise during DNA replication can be corrected by DNA polymerase proofreading or by post-replication mismatch repair (MMR). Inactivation of both mutation-avoidance systems results in extremely high mutability that can lead to error catastrophe 1,2 . High mutability and the likelihood of cancer can be caused by mutations and epigenetic changes that reduce MMR 3,4 . Hypermutability can also be caused by external factors that directly inhibit MMR. Identifying such factors has important implications for understanding the role of the environment in genome stability. We found that chronic exposure of yeast to environmentally relevant concentrations of cadmium, a known human carcinogen 5 , can result in extreme hypermutability. The mutation specificity along with responses in proofreading-deficient and MMR-deficient mutants indicate that cadmium reduces the capacity for MMR of small misalignments and base-base mismatches. In extracts of human cells, cadmium inhibited at least one step leading to mismatch removal. Together, our data show that a high level of genetic instability can result from environmental impediment of a mutation-avoidance system.To identify factors that might influence MMR, we used long homonucleotide runs in the yeast Saccharomyces cerevisiae as reporters of altered MMR capacity. Long homonucleotide runs and other microsatellites are at-risk motifs in MMR-deficient cells 6 . These motifs are sensitive reporters for low levels of MMR 7-10 and for MMR-deficient cancers 3,4,11 because the correction of frequently occurring spontaneous frameshift errors in long homonucleotide runs and microsatellites is accomplished primarily by MMR. Frameshifts in shorter runs and base substitutions are corrected by proofreading as well as by MMR 8,12 .We chose to test divalent cations as potential inhibitors of MMR because they can affect many enzymatic reactions. Several divalent cations that are also carcinogens can decrease the fidelity of DNA synthesis in vitro 13 . Thus, it seemed possible that these cations could also target mutation-avoidance systems in vivo. We found that chronic exposure to low, non-lethal doses of cadmium (CdCl 2 ) caused a substantial increase in mutability (as much as 2,000-fold) of long homonucleotide runs in the yeast gene LYS2 (Fig. 1a and Because the stability of long homonucleotide runs is extremely sensitive to reduction in MMR capacity, we proposed that MMR itself is a target of cadmium. Mutations that completely eliminate DNA-damage repair pathways have never been reported to cause such strong mutator effects as are caused by MMR defects in long homonucleotide runs and other microsatellites 3 . Our experiments with isogenic strains that lack base-excision repair (rad27-Δ16), nucleotide excision repair (rad1-Δ, data not shown) and double-strand break repair (rad52-Δ, data not shown) identified only moderate mutator effects as compared with those caused by defects in MMR. The hypothesis about MMR inhibition was supported by a comparison of the effects of cadm...