ADP-dependent DNA Strand Exchange by the Mutant
[P67G/E68A]RecA Protein
EVIDENCE FOR AN INVOLVEMENT OF ADP IN RecA PROTEIN-MEDIATED
BRANCH MIGRATION*
Sunil
Nayak
,
Eric L.
Hildebrand, and
Floyd R.
Bryant§
From the Department of Biochemistry, The Johns Hopkins University,
School of Public Health, Baltimore, Maryland 21205
We have prepared a mutant RecA protein in which
proline 67 and glutamic acid 68 in the NTP binding site were replaced
by a glycine and alanine residue, respectively. The [P67G/E68A]RecA protein catalyzes the single-stranded DNA-dependent
hydrolysis of ATP and is able to promote the standard
ATP-dependent three-strand exchange reaction between a
circular bacteriophage 
X174 (X) single-stranded DNA molecule and
a homologous linear X double-stranded (ds) DNA molecule (5.4 kilobase pairs). The strand exchange activity differs from that of the
wild type RecA protein, however, in that it is (i) completely inhibited
by an ATP regeneration system, and (ii) strongly stimulated by the
addition of high concentrations of ADP to the reaction solution. These
results indicate that the strand exchange activity of the
[P67G/E68A]RecA protein is dependent on the presence of both ATP and
ADP. The ADP dependence of the reaction is reduced or eliminated when
(i) a shorter linear X dsDNA fragment (1.1 kilobase pairs) is
substituted for the full-length linear X dsDNA substrate, or (ii)
the Mg2+ concentration is reduced to a level just
sufficient to complex the ATP present in the reaction solution. These
results indicate that it is the branch migration phase (and not the
initial pairing step) of the [P67G/E68A]RecA protein-promoted strand
exchange reaction that is dependent on ADP. It is likely that the
[P67G/E68A]RecA mutation has revealed a requirement for ADP that also
exists (but is not as readily apparent) in the strand exchange reaction
of the wild type RecA protein.
*
This work was supported by Grant GM36516 from the National
Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
These authors contributed equally to this work.
§
To whom correspondence should be addressed. Tel.: 410-955-3895;
Fax: 410-472-3378; E-mail: fbryant@jhsph.edu.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
