Article

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: 33.12). 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 · 3.36 Impact Factor
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    • "Given the importance of MMR to genome stability, redundant signals are to be expected. Candidate signals include the 3′-DNA ends used for chain elongation and/or PCNA (e.g., (Hombauer et al., 2011; Kleczkowska et al., 2001; Kunkel and Erie, 2005; Modrich and Lahue, 1996; Pavlov et al., 2003; Pluciennik et al., 2010; Tran et al., 1999; Umar et al., 1996 "
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    Molecular cell 04/2013; 50(3). DOI:10.1016/j.molcel.2013.03.017 · 14.46 Impact Factor
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    • "In addition, exonuclease 1 (Exo1), a Rad2 family exonuclease that hydrolyzes double stranded DNA (dsDNA) with a 5 –3 polarity, serves as an important component in the eukaryotic MMR excision step for removing the mismatched base [7] [8]. Much of the recent advance in bacterial MMR has been the demonstration of the nicking endonuclease activity of MutL from several MutH-less bacteria and determination of the structure of the MutL endonuclease domain [9] [10] [11] [12] [13] [14] [15]. "
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    DNA repair 03/2013; DOI:10.1016/j.dnarep.2013.02.002 · 3.36 Impact Factor
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