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RecA homology search is promoted by mechanical stress along the scanned duplex DNA.

ABSTRACT: A RecA-single-stranded DNA (RecA-ssDNA) filament searches a genome for sequence homology by rapidly binding and unbinding double-stranded DNA (dsDNA) until homology is found. We demonstrate that pulling on the opposite termini (3' and 5') of one of the two DNA strands in a dsDNA molecule stabilizes the normally unstable binding of that dsDNA to non-homologous RecA-ssDNA filaments, whereas pulling on the two 3', the two 5', or all four termini does not. We propose that the 'outgoing' strand in the dsDNA is extended by strong DNA-protein contacts, whereas the 'complementary' strand is extended by the tension on the base pairs that connect the 'complementary' strand to the 'outgoing' strand. The stress resulting from different levels of tension on its constitutive strands causes rapid dsDNA unbinding unless sufficient homology is present.

SUBMITTER: Danilowicz C

PROVIDER: S-EPMC3287184 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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RecA homology search is promoted by mechanical stress along the scanned duplex DNA.

Danilowicz Claudia C Feinstein Efraim E Conover Alyson A Coljee Vincent W VW Vlassakis Julea J Chan Yuen-Ling YL Bishop Douglas K DK Prentiss Mara M

Nucleic acids research 20111019 4

A RecA-single-stranded DNA (RecA-ssDNA) filament searches a genome for sequence homology by rapidly binding and unbinding double-stranded DNA (dsDNA) until homology is found. We demonstrate that pulling on the opposite termini (3' and 5') of one of the two DNA strands in a dsDNA molecule stabilizes the normally unstable binding of that dsDNA to non-homologous RecA-ssDNA filaments, whereas pulling on the two 3', the two 5', or all four termini does not. We propose that the 'outgoing' strand in th ...[more]

PMID: 22013164

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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

RecA homology search is promoted by mechanical stress along the scanned duplex DNA.

Publications

RecA homology search is promoted by mechanical stress along the scanned duplex DNA.

Similar Datasets

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