Transfer of recA protein from one polynucleotide to another. Kinetic evidence for a ternary intermediate during the transfer reaction.

J. P. Menetski, S. C. Kowalczykowski

Research output: Contribution to journalArticle

33 Scopus citations

Abstract

We have analyzed the transfer kinetics of recA protein from one polynucleotide to another by monitoring the change in fluorescence of a modified single-stranded M13 DNA, referred to as etheno M13 DNA, that accompanies recA protein dissociation. The observed rate of transfer is dependent on the concentration of competitor polynucleotide, polythymidylic acid (poly(dT]; increasing the poly(dT) concentration increases the rate of transfer. These data are inconsistent with the rate-limiting step in the transfer mechanism occurring by a simple dissociation process. Under certain conditions, the apparent rate constant displays plateau behavior at high poly(dT) concentrations. This result is indicative of transfer occurring through a ternary intermediate including etheno M13 DNA and poly(dT). The transfer reaction was found to occur through two kinetically distinct species of transferring recA protein X DNA complexes. The relative amounts of these two species was affected by both the MgCl2 and protein concentration, suggesting that the two kinetic components reflect different aggregation states of the recA protein X DNA complex. Because etheno M13 DNA and poly(dT) contain no complementary sequences, we have concluded that recA protein has the intrinsic ability to form a kinetic ternary intermediate with two separate single-stranded DNA molecules in the absence of homology.

Original languageEnglish (US)
Pages (from-to)2085-2092
Number of pages8
JournalJournal of Biological Chemistry
Volume262
Issue number5
StatePublished - Feb 15 1987
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry

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