Visualization of Eukaryotic DNA Mismatch Repair Reveals Distinct Recognition and Repair Intermediates

Ludwig Institute for Cancer Research, University of California School of Medicine, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0669, USA.
Cell (Impact Factor: 32.24). 11/2011; 147(5):1040-53. DOI: 10.1016/j.cell.2011.10.025
Source: PubMed

ABSTRACT DNA mismatch repair (MMR) increases replication fidelity by eliminating mispaired bases resulting from replication errors. In Saccharomyces cerevisiae, mispairs are primarily detected by the Msh2-Msh6 complex and corrected following recruitment of the Mlh1-Pms1 complex. Here, we visualized functional fluorescent versions of Msh2-Msh6 and Mlh1-Pms1 in living cells. We found that the Msh2-Msh6 complex is an S phase component of replication centers independent of mispaired bases; this localized pool accounted for 10%-15% of MMR in wild-type cells but was essential for MMR in the absence of Exo1. Unexpectedly, Mlh1-Pms1 formed nuclear foci that, although dependent on Msh2-Msh6 for formation, rarely colocalized with Msh2-Msh6 replication-associated foci. Mlh1-Pms1 foci increased when the number of mispaired bases was increased; in contrast, Msh2-Msh6 foci were unaffected. These findings suggest the presence of replication machinery-coupled and -independent pathways for mispair recognition by Msh2-Msh6, which direct formation of superstoichiometric Mlh1-Pms1 foci that represent sites of active MMR.

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    • "This led us to wonder if SWR-C might also modulate Exo1 activity in other DNA transactions. For example, Exo1 can contribute to DNA flap removal during Okazaki fragment maturation of the nascent lagging strand [6], and it can excise replication errors during post-replication mismatch repair (MMR) [7], especially mismatches generated during lagging strand replication by DNA polymerase ␦ (Pol ␦) and Pol ␣ [8] [9]. Furthermore, several studies indicate that nucleosome assembly can regulate mismatch repair at replication forks. "
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    ABSTRACT: The yeast SWR-C chromatin remodeling enzyme catalyzes chromatin incorporation of the histone variant H2A.Z which plays roles in transcription, DNA repair, and chromosome segregation. Dynamic incorporation of H2A.Z by SWR-C also enhances the ability of exonuclease I (Exo1) to process DNA ends during repair of double strand breaks. Given that Exo1 also participates in DNA replication and mismatch repair, here we test whether SWR-C influences DNA replication fidelity. We find that inactivation of SWR-C elevates the spontaneous mutation rate of a strain encoding a L612M variant of DNA polymerase (Pol) δ, with a single base mutation signature characteristic of lagging strand replication errors. However, this genomic instability does not solely result from reduced Exo1 function, because single base mutator effects are seen in both Exo1-proficient and Exo1-deficient pol3-L612M swr1Δ strains. The data are consistent with the possibility that incorporation of the H2A.Z variant by SWR-C may stimulate Exo1 activity, as well as enhance the fidelity of replication by Pol δ, the repair of mismatches generated by Pol δ, or both. Copyright © 2014 Elsevier B.V. All rights reserved.
    DNA Repair 11/2014; 25C:9-14. DOI:10.1016/j.dnarep.2014.10.010 · 3.11 Impact Factor
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    • "These proteins were confirmed to be enriched at nascent DNA in ESCs by western blot with specific antibodies (Figure 3A). The enrichment on MMR proteins at nascent DNA concurs with previous studies in yeast (26) and the more recent iPOND mass-spectrometry data on HEK-293T cells (10,11). These results suggest that DNA repair systems could be an integral part of the replication machinery across different cell types, although analysis of the mutation frequency (12) and total proteins levels between fibroblasts and ESCs (Supplementary Figure S5) indicate quantitative differences in MMR system activity in ESCs and somatic cells. "
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    ABSTRACT: Pluripotency of embryonic stem cells (ESCs) is maintained by transcriptional activities and chromatin modifying complexes highly organized within the chromatin. Although much effort has been focused on identifying genome-binding sites, little is known on their dynamic association with chromatin across cell divisions. Here, we used a modified version of the iPOND (isolation of proteins at nascent DNA) technology to identify a large protein network enriched at nascent DNA in ESCs. This comprehensive and unbiased proteomic characterization in ESCs reveals that, in addition to the core replication machinery, proteins relevant for pluripotency of ESCs are present at DNA replication sites. In particular, we show that the chromatin remodeller HDAC1–NuRD complex is enriched at nascent DNA. Interestingly, an acute block of HDAC1 in ESCs leads to increased acetylation of histone H3 lysine 9 at nascent DNA together with a concomitant loss of methylation. Consistently, in contrast to what has been described in tumour cell lines, these chromatin marks were found to be stable during cell cycle progression of ESCs. Our results are therefore compatible with a rapid deacetylation-coupled methylation mechanism during the replication of DNA in ESCs that may participate in the preservation of pluripotency of ESCs during replication.
    Nucleic Acids Research 05/2014; 42(11). DOI:10.1093/nar/gku374 · 9.11 Impact Factor
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    • "MSH2 can heterodimerize with MSH6 or MSH3 and can play a critical role in the recognition and signaling of MMR (38). However, the MSH2-MSH6 complex has also been reported to be an S phase component of replication centers independent of mispaired bases (39). Some nuclear foci formed by MSH2-MSH6 do not necessarily represent sites of active MMR (39), suggesting that MSH2 has a role beyond canonical MMR in vivo. "
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    ABSTRACT: Translesion DNA synthesis (TLS) can use specialized DNA polymerases to insert and/or extend nucleotides across lesions, thereby limiting stalled replication fork collapse and the potential for cell death. Recent studies have shown that monoubiquitinated proliferating cell nuclear antigen (PCNA) plays an important role in recruitment of Y-family TLS polymerases to stalled replication forks after DNA damage treatment. To explore the possible roles of other factors that regulate the ultraviolet (UV)-induced assembly of specialized DNA polymerases at arrested replication forks, we performed immunoprecipitation experiments combined with mass spectrometry and established that DNA polymerase kappa (Polκ) can partner with MSH2, an important mismatch repair protein associated with hereditary non-polyposis colorectal cancer. We found that depletion of MSH2 impairs PCNA monoubiquitination and the formation of foci containing Polκ and other TLS polymerases after UV irradiation of cells. Interestingly, expression of MSH2 in Rad18-deficient cells increased UV-induced Polκ and REV1 focus formation without detectable changes in PCNA monoubiquitination, indicating that MSH2 can regulate post-UV focus formation by specialized DNA polymerases in both PCNA monoubiquitination-dependent and -independent fashions. Moreover, we observed that MSH2 can facilitate TLS across cyclobutane pyrimidine dimers photoproducts in living cells, presenting a novel role of MSH2 in post-UV cellular responses.
    Nucleic Acids Research 09/2013; 41(22). DOI:10.1093/nar/gkt793 · 9.11 Impact Factor
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