NPE was depleted using pre-immune antibodies (lane 1), a mixture of Msh2 and Msh6 (A; lane 2), or Mlh1 antibodies (B; lane 2)

NPE was depleted using pre-immune antibodies (lane 1), a mixture of Msh2 and Msh6 (A; lane 2), or Mlh1 antibodies (B; lane 2). PCNA-interacting motif, thereby extending significantly the temporal window permissive to strand-specific MMR. Our data identify DNA-bound PCNA as the signal that enables strand discrimination after the disappearance of strand discontinuities, and uncover a novel role of MutS in the retention of the post-replicative MMR capability. DOI: MMR system can also correct replication errors through a methylation-independent mechanism, where strand discontinuities can substitute for GATC methylation both in vivo and in vitro?(Laengle-Rouault et al., 1986; Lahue et al., 1987; 1989). Eukaryotic MMR is directed by strand discontinuities such as nicks or gaps in vitro?(Holmes et al., 1990; Thomas et al., 1991). Two MutS heterodimers, MutS (Msh2-Msh6) and MutS (Msh2-Msh3) recognize replication errors; MutS has a biased preference for base-base mismatches and small insertion/deletion loops (IDLs), while MutS preferentially recognizes large IDLs (Iyer et al., 2006; Jiricny, 2013; Kunkel and Erie, 2015). After mismatch binding, MutS/ are converted into closed clamp-like forms, by which they can translocate along DNA. They then recruit the latent nicking-endonuclease MutL (Mlh1-Pms2 in vertebrates and Mlh1-Pms1 in yeast) to initiate the removal of the error-carrying DNA strand. Two other eukaryotic MutL homologs, MutL (Mlh1-Pms1 in vertebrates and Mlh1-Mlh2 in yeast) and MutL (Mlh1-Mlh3) are suggested to play minor roles in somatic MMR (Jiricny, 2013; Campbell et al., 2014). Successful reconstitutions of eukaryotic MMR have shown that MutS, or MutS, and MutL, the Proliferating Cell Nuclear Antigen (PCNA) sliding clamp, the clamp loader Replication Factor C (RFC), the Exo1 Clioquinol exonuclease, and the DNA synthesis components promote MMR when a strand discontinuity is present (Genschel and Modrich, 2003; Dzantiev et al., 2004; Constantin et al., 2005; Zhang et al., 2005). A strand discontinuity, which can occur on either 5- or 3-side of the mismatch, activates MutL through a MutS- and PCNA-dependent mechanism to induce successive rounds of nicking on the error-carrying strand (Kadyrov et al., 2006; 2007; Pluciennik et al., 2010). Single-strand DNA termini such as 5-ends of the Okazaki fragments serve as entry points for Exo1 and strand discrimination signals in vivo?as well (Pavlov et al., 2003; Nick McElhinny et al., 2010; Liberti et al., 2013; Lujan et al., 2014; Liu et al., 2015). Recent studies have revealed that ribonucleotides embedded by replicative DNA polymerases serve as strand signals for MMR in vitro and contribute to Clioquinol a sub-fraction of leading strand MMR in vivo, after converted into single-strand gaps through RNaseH2-dependent ribonucleotide excision repair (Ghodgaonkar et al., 2013; Lujan et al., 2013). PCNA has also been presumed to be the strand discrimination marker in eukaryotes (Umar et al., 1996; Chen et al., 1999; Pavlov et al., 2003; Dzantiev et al., 2004; Kadyrov et al., 2006; Pluciennik et al., 2010; Hombauer et al., 2011b; Pe?a-Diaz and Jiricny, 2012; Georgescu et al., 2015; Kunkel and Erie, 2015). PCNA is a ring-shaped homo-trimer that supports various DNA transactions including DNA replication and Clioquinol repair (Georgescu et al., 2015). PCNA is loaded onto DNA from the template-primer junction by RFC, and likely unloaded by an RFC-like complex containing Elg1 after the completion of DNA synthesis (Kubota et al., 2013; Rabbit Polyclonal to IL18R 2015). Since its DNA binding is asymmetric with respect to polarities of the parental and daughter strands, DNA-bound PCNA can hold information for the newly synthesized strand (Bowman et al., 2004; Georgescu et al., 2015). PCNA plays an essential role in an early MMR step that precedes degradation of the error-carrying strand (Umar et al., 1996). PCNA loaded from a strand discontinuity induces strand-specific, mismatch- and MutS-dependent activation of the?MutL endonuclease (Kadyrov et al., 2006; 2007; Pluciennik et al., 2010). When PCNA is loaded onto closed circular DNA without defined orientation, it induces unbiased nicking on both DNA strands (Pluciennik et al., 2010; 2013). These findings have led to a proposal that orientation of DNA-bound PCNA is a critical determinant for the nicking specificity of MutL. In addition to its proposed role in strand discrimination, PCNA is also involved in multiple steps of MMR. Numbers of PCNA mutants in yeast exhibit mutator phenotypes that are epistatic to MMR mutations (Johnson et al., 1996; Umar et al., 1996; Chen et al., 1999; Amin et al., 2001; Lau et al., 2002; Goellner et al., 2014). It interacts with MutS/ through their PCNA-interacting peptide (PIP) motifs, which reside at the N-termini of Msh6 and Msh3 (Clark et al., 2000; Flores-Rozas et al., 2000; Kleczkowska Clioquinol et al., 2001). In both cases, the PIP motifs and the mispair binding domains are connected through long linkers, which are predicted to be disordered in yeast (Shell et al., 2007). Mutants of the PIP motifs in Msh6 and Msh3 show substantial but not complete reduction of the MMR activity, indicating that the PIP motif plays.