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Nucleic Acids Res. 2011 May;39(9):3781-91. doi: 10.1093/nar/gkr013. Epub 2011 Jan 18.

Single-molecule studies of the stringency factors and rates governing the polymerization of RecA on double-stranded DNA.

Author information

  • 1Department of Physics, Harvard University, Cambridge, MA, USA. feinst@post.harvard.edu

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 µM, 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.

© The Author(s) 2011. Published by Oxford University Press.

PMID:
21245047
[PubMed - indexed for MEDLINE]
PMCID:
PMC3089484
Free PMC Article

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