Properties of recA441 protein-catalyzed DNA strand exchange can be attributed to an enhanced ability to compete with SSB protein.
We have investigated the recombinase activity of recA441 protein by comparing its in vitro DNA strand exchange activity to that of wild-type recA protein. Consistent with its proficiency in recombination in vivo, recA441 protein is able to catalyze the in vitro exchange of a circular single-stranded DNA molecule for a homologous strand in a linear double-stranded DNA molecule. Under conditions optimal for wild-type recA protein, the rates of joint molecule formation are the same for the two recA proteins, but the wild-type protein converts these intermediate species to gapped circular heteroduplex DNA product molecules more rapidly than recA441 protein. In the recA441 protein reaction, joint molecules are instead converted to extensive homology-dependent DNA networks via presumed reinitiation reactions. Under some conditions, the DNA strand exchange activity of recA441 protein is enhanced relative to the wild-type. These conditions include when single-stranded DNA.SSB protein (where SSB is Escherichia coli single-stranded DNA-binding protein) complexes are formed prior to the addition of recA protein, at low magnesium ion concentration in the presence of spermidine, and at low ATP concentrations. Under the conditions examined, recA441 protein competes more effectively with SSB protein for DNA-binding sites; thus, the differences between the strand exchange activities of the wild-type and recA441 proteins can be attributed to this enhanced ability in SSB protein competition.
Lavery
P E
PE
Department of Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611.
Kowalczykowski
S C
SC
D000085:Acetates / Q000494:pharmacology; D019342:Acetic Acid; D001667:Binding, Competitive; D003090:Coliphages / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D011938:Rec A Recombinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D000085:Acetates; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D011938:Rec A Recombinases; D019342:Acetic Acid