On the role of ATP hydrolysis in RecA protein-mediated DNA strand exchange. I. Bypassing a short heterologous insert in one DNA substrate.
RecA protein promotes a substantial DNA strand exchange reaction in the presence of adenosine 5'-O-3-(thio)triphosphate (ATP gamma S) (Menetski, J.P., Bear, D.G., and Kowalczykowski, S.C. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 21-25), calling into question the role of ATP hydrolysis in the strand exchange reaction. Here, we demonstrate that the ATP gamma S-mediated reaction can go to completion when the duplex DNA substrate is only 1.3 kilobase pairs in length. The ATP gamma S-mediated reaction, however, is completely blocked by a 52-base pair heterologous insertion in either DNA substrate. This same barrier is readily bypassed when ATP replaces ATP gamma S. This indicates that at least one function of recA-mediated ATP hydrolysis is to bypass structural barriers in one or both DNA substrates during strand exchange. This suggests that ATP hydrolysis is directly coupled to the branch migration phase of strand exchange, not to promote strand exchange between homologous DNA substrates during recombination, but instead to facilitate the bypass of structural barriers likely to be encountered during recombinational DNA repair.
Kim
J I
JI
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706.
Cox
M M
MM
Inman
R B
RB
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology; D003090:Coliphages / Q000378:metabolism; D004270:DNA, Circular / Q000378:metabolism / Q000648:ultrastructure; D004277:DNA, Single-Stranded / Q000378:metabolism / Q000648:ultrastructure; D004279:DNA, Viral / Q000378:metabolism* / Q000648:ultrastructure; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008854:Microscopy, Electron; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism*; D013379:Substrate Specificity
D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D011938:Rec A Recombinases