RecA protein-promoted DNA strand exchange. Stable complexes of recA protein and single-stranded DNA formed in the presence of ATP and single-stranded DNA binding protein.

M M Cox,I R Lehman

doi:10.1016/s0021-9258(18)34363-1

Abstract

The recA protein of Escherichia coli promotes the complete exchange of strands between full length linear duplex and single-stranded circular DNA molecules. An early step in this reaction consists of the binding of recA protein to single-stranded DNA. In the presence of ATP and the single-stranded DNA binding protein, recA protein and single-stranded DNA interact to form a complex whose stability depends upon the single-stranded DNA binding protein. Duplex DNA is not required for complex formation. Subsequent steps occur within this complex which contains up to 1 recA protein monomer per 2 nucleotides of single-stranded DNA. Although several hundred ATPs are hydrolyzed per recA protein monomer, recA protein is not released from the complex at any step during or subsequent to strand exchange. The complex is kinetically competent with respect to both its rate of formation and its rate of reaction with homologous duplex DNA. It is therefore a significant intermediate in the overall reaction pathway. These results have served as the basis for an expanded model for recA protein-promoted strand exchange.

Highlights

TherecAprotein of Escherichia coli promotesthe pled vectorial process in which the chemical energy of ATP is complete exchange of strands between full length lin- utilized to bring about a unidirectional branch migration
Duplex DNA is protein-promoted strand exchange, we have investigated the not required for complex formation
To reduce the salt content in the pyruvate kinase preparation, an aliquot of the ammosingle-stranded and linear duplex DNAs has been informative [3, 5,6,7,8]. recA protein-promoted strand exchange proceeds in two kinetically distinguishable phases (3, 7 ) .The first, formation of a short heteroduplex region, is relatively fast, proceeds in the presence of the nonhydrolyzable ATP analog adenosine 5'-0-(thiotriphosphate), and can nium sulfate suspension was centrifuged and the pellet was resuspended in reaction buffer to the original volume

Summary

EXPERIMENTAL PROCEDURES

Way.These results haveserved as the basis for an expanded model for recA protein-promoted strandexchange. RecA protein-promoted strand exchange proceeds in two kinetically distinguishable phases (3, 7 ) .The first, formation of a short heteroduplex region (a D-loop), is relatively fast, proceeds in the presence of the nonhydrolyzable ATP analog adenosine 5'-0-(thiotriphosphate), and can nium sulfate suspension was centrifuged and the pellet was resuspended in reaction buffer to the original volume. S S DNA has been described ( 3 ) .Supercoiled SV40 DNA was a gift from Michael Fromm of this department. It was linearized by cleavage with the restriction endonuclease H p a 11, extracted with phenol. Metric, rather than catalytic, amounts of recA protein are required and thereaction is strongly stimulated by SSB.'

Methods

RESULTS

A ss ss

Findings

DISCUSSION