Single-molecule Studies of the Stringency Factors and Rates Governing the Polymerization of RecA on Double-stranded DNA

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Single-molecule Studies of the Stringency Factors and Rates Governing the Polymerization of RecA on Double-stranded DNA

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Title: Single-molecule Studies of the Stringency Factors and Rates Governing the Polymerization of RecA on Double-stranded DNA
Author: Feinstein, Efraim; Conover, Alyson; Gunaratne, Ruwan; Danilowicz, Claudia; Kleckner, Nancy Elizabeth; Prentiss, Mara

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Citation: Feinstein, Efraim, Claudia Danilowicz, Alyson Conover, Ruwan Gunaratne, Nancy Kleckner, and Mara Prentiss. 2011. Single-molecule studies of the stringency factors and rates governing the polymerization of RecA on double-stranded DNA. Nucleic Acids Research 39(9): 3781-3791.
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Abstract: RecA is a key protein in homologous recombination. During recombination, one single-stranded DNA (ssDNA) bound to site I in RecA exchanges Watson-Crick pairing with a sequence-matched ssDNA that was part of a double-stranded DNA molecule (dsDNA) bound to site II in RecA. After strand exchange, heteroduplex dsDNA is bound to site I. In vivo, direct polymerization of RecA on dsDNA through site I does not occur, though it does in vitro. The mechanisms underlying the difference have been unclear. We use single-molecule experiments to decouple the two steps involved in polymerization: nucleation and elongation. We find that elongation is governed by a fundamental clock that is insensitive to force and RecA concentration from 0.2 and 6\(\mu\)M, though rates depend on ionic conditions. Thus, we can probe nucleation site stability by creating nucleation sites at high force and then measuring elongation as a function of applied force. We find that in the presence of ATP hydrolysis a minimum force is required for polymerization. The minimum force decreases with increasing RecA or ATP concentrations. We propose that force reduces the off-rate for nucleation site binding and that nucleation site stability is the stringency factor that prevents in vivo polymerization.
Published Version: doi://10.1093/nar/gkr013
Other Sources: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3089484/pdf/
Terms of Use: This article is made available under the terms and conditions applicable to Other Posted Material, as set forth at http://nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA
Citable link to this page: http://nrs.harvard.edu/urn-3:HUL.InstRepos:11213360
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