title: Activation of Saccharomyces cerevisiae Mlh1-Pms1 Endonuclease in a Reconstituted Mismatch Repair System.
abstract: Previous studies reported the reconstitution of an Mlh1-Pms1-independent 5' nick-directed mismatch repair (MMR) reaction using Saccharomyces cerevisiae proteins. Here we describe the reconstitution of a mispair-dependent Mlh1-Pms1 endonuclease activation reaction requiring Msh2-Msh6 (or Msh2-Msh3), proliferating cell nuclear antigen (PCNA), and replication factor C (RFC) and a reconstituted Mlh1-Pms1-dependent 3' nick-directed MMR reaction requiring Msh2-Msh6 (or Msh2-Msh3), exonuclease 1 (Exo1), replication protein A (RPA), RFC, PCNA, and DNA polymerase δ. Both reactions required Mg(2+) and Mn(2+) for optimal activity. The MMR reaction also required two reaction stages in which the first stage required incubation of Mlh1-Pms1 with substrate DNA, with or without Msh2-Msh6 (or Msh2-Msh3), PCNA, and RFC but did not require nicking of the substrate, followed by a second stage in which other proteins were added. Analysis of different mutant proteins demonstrated that both reactions required a functional Mlh1-Pms1 endonuclease active site, as well as mispair recognition and Mlh1-Pms1 recruitment by Msh2-Msh6 but not sliding clamp formation. Mutant Mlh1-Pms1 and PCNA proteins that were defective for Exo1-independent but not Exo1-dependent MMR in vivo were partially defective in the Mlh1-Pms1 endonuclease and MMR reactions, suggesting that both reactions reflect the activation of Mlh1-Pms1 seen in Exo1-independent MMR in vivo. The availability of this reconstituted MMR reaction should now make it possible to better study both Exo1-independent and Exo1-dependent MMR.
authors.affiliation: From the Ludwig Institute for Cancer Research.
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Smith
authors.affiliation: From the Ludwig Institute for Cancer Research.
authors.forename: Nikki
authors.initials: N
authors.lastname: Bowen
authors.affiliation: From the Ludwig Institute for Cancer Research.
authors.forename: William J
authors.initials: WJ
authors.lastname: Graham
authors.affiliation: From the Ludwig Institute for Cancer Research.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Goellner
authors.affiliation: From the Ludwig Institute for Cancer Research.
authors.forename: Anjana
authors.initials: A
authors.lastname: Srivatsan
authors.affiliation: From the Ludwig Institute for Cancer Research, the Department of Cellular and Molecular Medicine, Moores-UCSD Cancer Center, and the Institute of Genomic Medicine, University of California, San Diego School of Medicine, La Jolla, California 92093 rkolodner@ucsd.edu.
authors.forename: Richard D
authors.initials: RD
authors.lastname: Kolodner
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D002352:Carrier Proteins / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D053843:DNA Mismatch Repair / Q000187:drug effects; D004789:Enzyme Activation / Q000187:drug effects; D005799:Genes, Dominant; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D002413:Cations, Divalent; C088859:MLH1 protein, S cerevisiae; D050505:Mutant Proteins; C088858:PMS1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins
keywords: DNA recombination; DNA repair; DNA replication; Msh2-Msh6; exonuclease 1; genome instability; mutagenesis; proliferating cell nuclear antigen (PCNA); replication factor C (RFC); yeast genetics