title: Reconstitution of long and short patch mismatch repair reactions using Saccharomyces cerevisiae proteins.
abstract: A problem in understanding eukaryotic DNA mismatch repair (MMR) mechanisms is linking insights into MMR mechanisms from genetics and cell-biology studies with those from biochemical studies of MMR proteins and reconstituted MMR reactions. This type of analysis has proven difficult because reconstitution approaches have been most successful for human MMR whereas analysis of MMR in vivo has been most advanced in the yeast Saccharomyces cerevisiae. Here, we describe the reconstitution of MMR reactions using purified S. cerevisiae proteins and mispair-containing DNA substrates. A mixture of MutS homolog 2 (Msh2)-MutS homolog 6, Exonuclease 1, replication protein A, replication factor C-Δ1N, proliferating cell nuclear antigen and DNA polymerase δ was found to repair substrates containing TG, CC, +1 (+T), +2 (+GC), and +4 (+ACGA) mispairs and either a 5' or 3' strand interruption with different efficiencies. The Msh2-MutS homolog 3 mispair recognition protein could substitute for the Msh2-Msh6 mispair recognition protein and showed a different specificity of repair of the different mispairs whereas addition of MutL homolog 1-postmeiotic segregation 1 had no affect on MMR. Repair was catalytic, with as many as 11 substrates repaired per molecule of Exo1. Repair of the substrates containing either a 5' or 3' strand interruption occurred by mispair binding-dependent 5' excision and subsequent resynthesis with excision tracts of up to ~2.9 kb occurring during the repair of the substrate with a 3' strand interruption. The availability of this reconstituted MMR reaction now makes possible detailed biochemical studies of the wealth of mutations identified that affect S. cerevisiae MMR.
authors.affiliation: Ludwig Institute for Cancer Research, Departments of Medicine and Cellular and Molecular Medicine, Moores-University of California, San Diego Cancer Center, and Institute of Genomic Medicine, University of California San Diego School of Medicine, La Jolla, CA 92093.
authors.forename: Nikki
authors.initials: N
authors.lastname: Bowen
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Smith
authors.forename: Anjana
authors.initials: A
authors.lastname: Srivatsan
authors.forename: Smaranda
authors.initials: S
authors.lastname: Willcox
authors.forename: Jack D
authors.initials: JD
authors.lastname: Griffith
authors.forename: Richard D
authors.initials: RD
authors.lastname: Kolodner
mesh_terms: D002384:Catalysis; D053843:DNA Mismatch Repair / Q000235:genetics*; D004258:DNA Polymerase III / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000378:metabolism; D042822:Genomic Instability / Q000235:genetics*; D046529:Microscopy, Electron, Transmission; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D051718:MutS Homolog 2 Protein / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*
chemical_list: D017931:DNA Primers; D004271:DNA, Fungal; D046912:Multiprotein Complexes; D018809:Proliferating Cell Nuclear Antigen; D051716:Replication Protein A; D029701:Saccharomyces cerevisiae Proteins; D004258:DNA Polymerase III; D005090:Exodeoxyribonucleases; C028450:exodeoxyribonuclease I; D051718:MutS Homolog 2 Protein
keywords: DNA replication fidelity; cancer; genome instability; mutagenesis; mutator phenotype