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

 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 Note: Order does not necessarily reflect citation order of authors. 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. Full Text & Related Files: 3089484.pdf (5.445Mb; PDF) 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 Downloads of this work: