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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 MutS? and MutL? as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutS? as it searches for DNA lesions, MutL? as it searches for lesion-bound MutS?, and the MutS?/MutL? complex as it scans the flanking DNA. We also show that MutL? undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutS?, but this activity is suppressed upon association with MutS?, 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 MutS? and MutL?, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA.

SUBMITTER: Gorman J

PROVIDER: S-EPMC3494904 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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

Gorman Jason J Wang Feng F Redding Sy S Plys Aaron J AJ Fazio Teresa T Wind Shalom S Alani Eric E EE Greene Eric C EC

Proceedings of the National Academy of Sciences of the United States of America 20120924 45

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 visu ...[more]

PMID: 23012240

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Project description:DNA breaks can be repaired with high fidelity by homologous recombination. A ubiquitous protein that is essential for this DNA template-directed repair is RecA. After resection of broken DNA to produce single-stranded DNA (ssDNA), RecA assembles on this ssDNA into a filament with the unique capacity to search and find DNA sequences in double-stranded DNA (dsDNA) that are homologous to the ssDNA. This homology search is vital to recombinational DNA repair, and results in homologous pairing and exchange of DNA strands. Homologous pairing involves DNA sequence-specific target location by the RecA-ssDNA complex. Despite decades of study, the mechanism of this enigmatic search process remains unknown. RecA is a DNA-dependent ATPase, but ATP hydrolysis is not required for DNA pairing and strand exchange, eliminating active search processes. Using dual optical trapping to manipulate DNA, and single-molecule fluorescence microscopy to image DNA pairing, we demonstrate that both the three-dimensional conformational state of the dsDNA target and the length of the homologous RecA-ssDNA filament have important roles in the homology search. We discovered that as the end-to-end distance of the target dsDNA molecule is increased, constraining the available three-dimensional (3D) conformations of the molecule, the rate of homologous pairing decreases. Conversely, when the length of the ssDNA in the nucleoprotein filament is increased, homology is found faster. We propose a model for the DNA homology search process termed 'intersegmental contact sampling', in which the intrinsic multivalent nature of the RecA nucleoprotein filament is used to search DNA sequence space within 3D domains of DNA, exploiting multiple weak contacts to rapidly search for homology. Our findings highlight the importance of the 3D conformational dynamics of DNA, reveal a previously unknown facet of the homology search, and provide insight into the mechanism of DNA target location by this member of a universal family of proteins.

Dataset Information

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

Publications

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

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