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, Floyd Ransom Bryant

Research output: Contribution to journalArticle

Abstract

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 φX174 (φX) single-stranded DNA molecule and a homologous linear φX 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 φX dsDNA fragment (1.1 kilobase pairs) is substituted for the full-length linear φX 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.

Original languageEnglish (US)
Pages (from-to)14933-14938
Number of pages6
JournalJournal of Biological Chemistry
Volume276
Issue number18
DOIs
StatePublished - May 4 2001

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Rec A Recombinases
Adenosine Diphosphate
Adenosine Triphosphate
DNA
Single-Stranded DNA
Molecules
Bacteriophages
Mutant Proteins
Proline
Alanine
Glycine
Regeneration
Glutamic Acid
Hydrolysis
Ion exchange
Binding Sites
Mutation
Substrates

ASJC Scopus subject areas

  • Biochemistry

Cite this

ADP-dependent DNA strand exchange by the mutant [P67G/E68A]RecA protein. Evidence for an involvement of ADP in RecA protein-mediated branch migration. / Nayak, Sunil; Hildebrand, Eric L.; Bryant, Floyd Ransom.

In: Journal of Biological Chemistry, Vol. 276, No. 18, 04.05.2001, p. 14933-14938.

Research output: Contribution to journalArticle

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