title: Reconstitution ofSaccharomyces cerevisiaeDNA polymerase ε-dependent mismatch repair with purified proteins.
abstract: Mammalian andSaccharomyces cerevisiaemismatch repair (MMR) proteins catalyze two MMR reactions in vitro. In one, mispair binding by either the MutS homolog 2 (Msh2)-MutS homolog 6 (Msh6) or the Msh2-MutS homolog 3 (Msh3) stimulates 5' to 3' excision by exonuclease 1 (Exo1) from a single-strand break 5' to the mispair, excising the mispair. In the other, Msh2-Msh6 or Msh2-Msh3 activate the MutL homolog 1 (Mlh1)-postmeiotic segregation 1 (Pms1) endonuclease in the presence of a mispair and a nick 3' to the mispair, to make nicks 5' to the mispair, allowing Exo1 to excise the mispair. DNA polymerase δ (Pol δ) is thought to catalyze DNA synthesis to fill in the gaps resulting from mispair excision. However, colocalization of theS. cerevisiaemispair recognition proteins with the replicative DNA polymerases during DNA replication has suggested that DNA polymerase ε (Pol ε) may also play a role in MMR. Here we describe the reconstitution of Pol ε-dependent MMR usingS. cerevisiaeproteins. A mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze both short-patch and long-patch 5' nick-directed MMR of a substrate containing a +1 (+T) mispair. When the substrate contained a nick 3' to the mispair, a mixture of Msh2-Msh6 (or Msh2-Msh3), Exo1, RPA, RFC-Δ1N, PCNA, and Pol ε was found to catalyze an MMR reaction that required Mlh1-Pms1. These results demonstrate that Pol ε can act in eukaryotic MMR in vitro.
authors.affiliation: Ludwig Institute for Cancer Research, University of California School of Medicine, La Jolla, CA 92093-0669.
authors.forename: Nikki
authors.initials: N
authors.lastname: Bowen
authors.affiliation: Ludwig Institute for Cancer Research, University of California School of Medicine, La Jolla, CA 92093-0669; rkolodner@ucsd.edu.|Department of Cellular and Molecular Medicine, University of California School of Medicine, La Jolla, CA 92093-0669.|Moores-University of California San Diego Cancer Center, University of California School of Medicine, La Jolla, CA 92093-0669.|Institute of Genomic Medicine, University of California School of Medicine, La Jolla, CA 92093-0669.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Kolodner
mesh_terms: D053843:DNA Mismatch Repair; D004268:DNA-Binding Proteins / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D000070957:MutL Protein Homolog 1 / Q000378:metabolism; D000070956:MutL Proteins / Q000378:metabolism; D051718:MutS Homolog 2 Protein / Q000378:metabolism; D000074081:MutS Homolog 3 Protein / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D000097462:DNA Polymerase theta
chemical_list: D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D051718:MutS Homolog 2 Protein; D000074081:MutS Homolog 3 Protein; D029701:Saccharomyces cerevisiae Proteins; D000097462:DNA Polymerase theta; C088859:MLH1 protein, S cerevisiae; C081696:MSH3 protein, S cerevisiae; C098790:MSH6 protein, S cerevisiae; C088858:PMS1 protein, S cerevisiae; C028450:exodeoxyribonuclease I; C489751:MSH2 protein, S cerevisiae
keywords: DNA excision; DNA repair; DNA replication fidelity; genome instability; mutator phenotype