Single-molecule imaging reveals target-search mechanisms during DNA mismatch repair

Departments of Biological Sciences, Biochemistry and Molecular Biophysics, and Chemistry, and Howard Hughes Medical Institute, Columbia University, New York, NY, 10032.
Proceedings of the National Academy of Sciences (Impact Factor: 9.81). 09/2012; 109(45). DOI: 10.1073/pnas.1211364109
Source: PubMed

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.

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    • "The nucleoid-association of Pol1 and Ligase suggests short-lived nonspecific protein–DNA interactions that might be part of a facilitated diffusion process to search for lesions. In vitro studies showed sliding on stretched DNA for a range of DNA-binding and repair proteins including p53 [71], MutS/MutL [72], oxoguanine glycosylase [73], and UvrAB nucleotide excision repair complexes [74]. On the other hand, E. coli RNA polymerase appeared to encounter promoters by direct collision without significant sliding [75]. "
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