title: Single-molecule imaging reveals target-search mechanisms during DNA mismatch repair.
abstract: The ability of proteins to locate specific targets among a vast excess of nonspecific DNA is a fundamental theme in biology. Basic principles governing these search mechanisms remain poorly understood, and no study has provided direct visualization of single proteins searching for and engaging target sites. Here we use the postreplicative mismatch repair proteins MutSα and MutLα as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutSα as it searches for DNA lesions, MutLα as it searches for lesion-bound MutSα, and the MutSα/MutLα complex as it scans the flanking DNA. We also show that MutLα undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutSα, but this activity is suppressed upon association with MutSα, ensuring that MutS/MutL remains associated with the damage-bearing strand while scanning the flanking DNA. Our findings highlight a hierarchy of lesion- and ATP-dependent transitions involving both MutSα and MutLα, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY 10032, USA.
authors.forename: Jason
authors.initials: J
authors.lastname: Gorman
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.forename: Sy
authors.initials: S
authors.lastname: Redding
authors.forename: Aaron J
authors.initials: AJ
authors.lastname: Plys
authors.forename: Teresa
authors.initials: T
authors.lastname: Fazio
authors.forename: Shalom
authors.initials: S
authors.lastname: Wind
authors.forename: Eric E
authors.initials: EE
authors.lastname: Alani
authors.forename: Eric C
authors.initials: EC
authors.lastname: Greene
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D053843:DNA Mismatch Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D004058:Diffusion; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008954:Models, Biological; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D021381:Protein Transport; D013379:Substrate Specificity
chemical_list: C500929:MutLalpha protein, human; D000255:Adenosine Triphosphate; D004247:DNA; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; D045643:DNA Repair Enzymes