title: Engineered disulfide-forming amino acid substitutions interfere with a conformational change in the mismatch recognition complex Msh2-Msh6 required for mismatch repair.
abstract: ATP binding causes the mispair-bound Msh2-Msh6 mismatch recognition complex to slide along the DNA away from the mismatch, and ATP is required for the mispair-dependent interaction between Msh2-Msh6 and Mlh1-Pms1. It has been inferred from these observations that ATP induces conformational changes in Msh2-Msh6; however, the nature of these conformational changes and their requirement in mismatch repair are poorly understood. Here we show that ATP induces a conformational change within the C-terminal region of Msh6 that protects the trypsin cleavage site after Msh6 residue Arg(1124). An engineered disulfide bond within this region prevented the ATP-driven conformational change and resulted in an Msh2-Msh6 complex that bound mispaired bases but could not form sliding clamps or bind Mlh1-Pms1. The engineered disulfide bond also reduced mismatch repair efficiency in vivo, indicating that this ATP-driven conformational change plays a role in mismatch repair.
authors.affiliation: Ludwig Institute for Cancer Research, Department of Medicine, Moores-University of California San Diego Cancer Center, and Institute of Genomic Medicine, University of California School of Medicine, San Diego, La Jolla, California 92093-0669, USA.
authors.forename: Victoria V
authors.initials: VV
authors.lastname: Hargreaves
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Putnam
authors.forename: Richard D
authors.initials: RD
authors.lastname: Kolodner
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000596:Amino Acids / Q000737:chemistry*; D020137:Base Pair Mismatch; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000494:pharmacology; D053843:DNA Mismatch Repair; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D006868:Hydrolysis; D051718:MutS Homolog 2 Protein / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D014357:Trypsin / Q000737:chemistry
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004220:Disulfides; C094443:G-T mismatch-binding protein; D000255:Adenosine Triphosphate; D014357:Trypsin; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein; D010100:Oxygen