Stable DNA heteroduplex formation catalyzed by the Escherichia coli RecA protein in the absence of ATP hydrolysis.
A question remaining to be answered about RecA protein function concerns the role of ATP hydrolysis during the DNA-strand-exchange reaction. In this paper we describe the formation of joint molecules in the absence of ATP hydrolysis, using adenosine 5'-[gamma-thio]triphosphate (ATP[gamma S]) as nucleotide cofactor. Upon the addition of double-stranded DNA, the ATP[gamma S]-RecA protein-single-stranded DNA presynaptic complexes can form homologously paired molecules that are stable after deproteinization. Formation of these joint molecules requires both homology and a free homologous end, suggesting that they are plectonemic in nature. This reaction is very sensitive to magnesium ion concentration, with a maximum rate and extent observed at 4-5 mM magnesium acetate. Under these conditions, the average length of heteroduplex DNA within the joint molecules is 2.4-3.4 kilobase pairs. Thus, RecA protein can form extensive regions of heteroduplex DNA in the presence of ATP[gamma S], suggesting that homologous pairing and the exchange of the DNA molecules can occur without ATP hydrolysis. A model for the RecA protein-catalyzed DNA-strand-exchange reaction that incorporates these results and its relevance to the mechanisms of eukaryotic recombinases are presented.
Department of Molecular Biology, Northwestern University Medical School, Chicago, IL 60611.
J P
JP
Menetski
D G
DG
Bear
S C
SC
Kowalczykowski
D000085:Acetates / Q000494:pharmacology; D019342:Acetic Acid; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D008961:Models, Structural; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism*
D000085:Acetates; D004269:DNA, Bacterial; D009692:Nucleic Acid Heteroduplexes; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D019342:Acetic Acid