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.

1 Follower
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "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 "
    [Show abstract] [Hide abstract]
    ABSTRACT: To maintain genome stability, mismatch repair of nuclear DNA replication errors must be directed to the nascent strand, likely by DNA ends and PCNA. Here we show that the efficiency of mismatch repair in Saccharomyces cerevisiae is reduced by inactivating RNase H2, which nicks DNA containing ribonucleotides incorporated during replication. In strains encoding mutator polymerases, this reduction is preferential for repair of mismatches made by leading-strand DNA polymerase ε as compared to lagging-strand DNA polymerase δ. The results suggest that RNase-H2-dependent processing of ribonucleotides transiently present in DNA after replication may direct mismatch repair to the continuously replicated nascent leading strand.
    Molecular cell 04/2013; 50(3). DOI:10.1016/j.molcel.2013.03.017 · 14.46 Impact Factor
  • Source
    • "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]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The most important system for correcting replication errors that survive the built in editing system of DNA polymerase is the mismatch repair (MMR) system. We have identified a novel mutator strain yycJ in Bacillus anthracis. Mutations in the yycJ gene result in a spontaneous mutator phenotype with a mutational frequency and specificity comparable to that of MMR-deficient strains such as those with mutations in mutL or mutS. YycJ was annotated as a metallo-β-lactamase (MβL) super family member with unknown activity. In this study we carried out a biochemical characterization of YycJ and demonstrated that a recombinant YycJ protein possesses a 5'-3' exonuclease activity at the 5' termini and at nicks of double-stranded DNA. This activity requires a divalent metal cofactor Mn(2+) and is stimulated by 5'-phosphate ends of duplex DNA. The mutagenesis of conserved amino acid residues revealed that in addition to the five MβL family conserved motifs, YycJ appears to have its specific motifs that can be used to distinguish YycJ from other closely related MβL family members. A phylogenetic survey showed that putative YycJ homologs are present in several bacterial phyla as well as in members of the Methanomicrobiales and Thermoplasmales from Archaea. We propose that YycJ represents a new group of MβL fold exonucleases, which is likely to act in the recognition of MMR entry point and subsequent removal of the mismatched base in certain MutH-less bacterial species.
    DNA repair 03/2013; DOI:10.1016/j.dnarep.2013.02.002 · 3.36 Impact Factor
Show more