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 R Bryant

doi:10.1074/jbc.m100470200

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 phiX174 (phiX) single-stranded DNA molecule and a homologous linear phiX 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 phiX dsDNA fragment (1.1 kilobase pairs) is substituted for the full-length linear phiX dsDNA substrate, or (ii) the Mg(2+) 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.

Highlights

We have prepared a mutant RecA protein in which proline and glutamic acid in the NTP binding site were replaced by a glycine and alanine residue, respectively
The strand exchange activity differs from that of the wild type RecA protein, 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
Strand Exchange Activity of the [P67G/E68A]RecA Protein Using a Shorter Linear dsDNA Substrate— the time required for the appearance of the fully exchanged circular dsDNA product in the [P67G/E68A]RecA protein-promoted strand exchange reaction was much greater than that for the wild type RecA protein, the partially exchanged DNA intermediates appeared to form at similar rates with the two proteins (Fig. 3)

Summary

Introduction

We have prepared a mutant RecA protein in which proline and glutamic acid in the NTP binding site were replaced by a glycine and alanine residue, respectively. When the starting nucleotide pool contained 0% ADP, the fully exchanged circular dsDNA products were detected ϳ4 h after the reaction was initiated, consistent with the results described above (Fig. 3).

Results

Conclusion