Activation of a recombinase-deficient mutant recA protein with alternate nucleoside triphosphate cofactors.
We recently described two mutant recA proteins, (G160N)recA and (H163A)recA, which have full single-stranded DNA-dependent ATP hydrolysis activity but which are unable to promote the ATP-dependent strand exchange reaction under standard reaction conditions (pH 7.5). These mutant proteins, however, are able to promote strand exchange at pH 6.0 to 6.8. Here we show that this activation correlates with a pH-dependent decrease in the S0.5 value for ATP, with the (H163A)recA protein becoming active in strand exchange at pH values where the S0.5(ATP) decreases below 100 microM. We also show that the (H163A)recA protein is active in strand exchange over the range of pH 6.0-8.2 if dATP (or ddATP) is used in place of ATP as a cofactor; dATP is hydrolyzed by (H163A)recA protein at the same rate as ATP but has an S0.5 value lower than 100 microM across this pH range. These results are discussed with regard to the general significance of the S0.5 value in determining whether a nucleoside triphosphate will be able to stabilize the recA-single-stranded DNA filament in the strand exchange active conformational state.
Meah
Y S
YS
Department of Biochemistry, Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205.
Bryant
F R
FR
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D004249:DNA Damage; D004254:DNA Nucleotidyltransferases / Q000235:genetics* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D006868:Hydrolysis; D019426:Integrases; D007700:Kinetics; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism*; D045522:Recombinases
D004277:DNA, Single-Stranded; D003838:Deoxyadenine Nucleotides; D045522:Recombinases; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D004254:DNA Nucleotidyltransferases; D019426:Integrases; D011938:Rec A Recombinases; C115006:integron integrase IntI1