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: 9.67). 09/2012; 109(45). DOI: 10.1073/pnas.1211364109
Source: PubMed


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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    • "In contrast to the metastable and dynamic nature of the TRF protein binding to telomeric sequences (Figure 5), other systems characterized by single-molecule imaging show long-lived stable binding to specific sequences. For example, the mismatch repair protein, MutSα binds to a mismatch (+ADP) with a half-life of 9.6 ± 1.5 min (36); and the average lifetime of the Type III restriction enzyme EcoP15I on DNA with specific binding sites was ∼180 s (38). The primary differences between these systems are the target DNA sites. "
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    ABSTRACT: Homologous recombination (HR) and mismatch repair (MMR) are inextricably linked. HR pairs homologous chromosomes before meiosis I and is ultimately responsible for generating genetic diversity during sexual reproduction. HR is initiated in meiosis by numerous programmed DNA double-strand breaks (DSBs; several hundred in mammals). A characteristic feature of HR is the exchange of DNA strands, which results in the formation of heteroduplex DNA. Mismatched nucleotides arise in heteroduplex DNA because the participating parental chromosomes contain nonidentical sequences. These mismatched nucleotides may be processed by MMR, resulting in nonreciprocal exchange of genetic information (gene conversion). MMR and HR also play prominent roles in mitotic cells during genome duplication; MMR rectifies polymerase misincorporation errors, whereas HR contributes to replication fork maintenance, as well as the repair of spontaneous DSBs and genotoxic lesions that affect both DNA strands. MMR suppresses HR when the heteroduplex DNA contains excessive mismatched nucleotides, termed homeologous recombination. The regulation of homeologous recombination by MMR ensures the accuracy of DSB repair and significantly contributes to species barriers during sexual reproduction. This review discusses the history, genetics, biochemistry, biophysics, and the current state of studies on the role of MMR in homologous and homeologous recombination from bacteria to humans. Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.
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