Dissociation pathway for recA nucleoprotein filaments formed on linear duplex DNA.
recA protein forms stable filaments on duplex DNA at low pH. When the pH is shifted above 6.8, recA protein remains stably bound to nicked circular DNA, but not to linear DNA. Dissociation of recA protein from linear duplex DNA proceeds to a non-zero endpoint. The kinetics and final extent of dissociation vary with several experimental parameters. The instability on linear DNA is most readily explained by a progressive unidirectional dissociation of recA protein from one end of the filament. Dissociation of recA protein from random points in the filament is eliminated as a possible mechanism by several observations: (1) the requirement for a free end; (2) the inverse and linear dependence of the rate of dissociation on DNA length (at constant DNA base-pair concentration); and (3) the kinetics of exposure of a restriction endonuclease site in the middle of the DNA. Evidence against another possible mechanism, ATP-mediated translocation of the filament along the DNA, is provided by a novel effect of the non-hydrolyzable ATP analog, ATP gamma S, which generally induces recA protein to bind any DNA tightly and completely inhibits ATP hydrolysis. We find that very low, sub-saturating levels of ATP gamma S completely stabilize the filament, while most of the ATP hydrolysis continues. If these levels of ATP gamma S are introduced after dissociation has commenced, further dissociation is blocked, but re-association does not occur. These observations are inconsistent with movement of recA protein along DNA that is tightly coupled to ATP hydrolysis. The recA nucleoprotein filament is polar and the protein binds the two strands asymmetrically, polymerizing mainly in the 5' to 3' direction on the initiating strand of a single-stranded DNA tailed duplex molecule. A model consistent with these results is presented.
Lindsley
J E
JE
Department of Biochemistry, College of Agriculture and Life Sciences, University of Wisconsin-Madison 53706.
Cox
M M
MM
D000255:Adenosine Triphosphate / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004278:DNA, Superhelical / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009698:Nucleoproteins / Q000378:metabolism*; D011938:Rec A Recombinases / Q000378:metabolism*
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004278:DNA, Superhelical; D009698:Nucleoproteins; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes