Biochemical events essential to the recombination activity of Escherichia coli RecA protein. I. Properties of the mutant RecA142 protein.
We have characterized the biochemical properties of Escherichia coli RecA142 protein, the product of a recA allele that is phenotypically defective in genetic recombination. In vitro, this mutant RecA protein is totally defective in DNA heteroduplex formation. Despite this defect, RecA142 protein is not deficient in all other biochemical activities. RecA142 protein is proficient in single-strand (ss) DNA binding ability, ssDNA-dependent ATPase activity, and DNA-free self-association (although the first 2 properties show a greater sensitivity to NaCl concentration than does the wild-type protein). However, RecA142 protein is deficient in four properties: (1) its ssDNA-dependent ATPase activity is completely inhibited by ssDNA binding (SSB) protein, demonstrating that RecA142 protein is unable to compete effectively with SSB protein for ssDNA binding sites; (2) it is unable to promote the coaggregation of ssDNA and double-strand (ds) DNA; (3) its M13 dsDNA-dependent ATPase activity is attenuated to approximately 5% of the level of the wild-type protein; (4) it is unable fully to develop characteristics of the high-affinity ssDNA-binding state that is normally induced by ATP. The first three deficiencies correspond to defects in the presynaptic, synaptic and postsynaptic steps of the in vitro DNA strand exchange reaction, respectively; the fourth is the likely fundamental basis for defects 1 and 3. Therefore, one or more of these properties must be important to both the in vitro and in vivo processes.
Kowalczykowski
S C
SC
Department of Molecular Biology, Northwestern University Medical School, Chicago, IL 60611.
Burk
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DL
Krupp
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RA
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D004269:DNA, Bacterial / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006868:Hydrolysis; D009154:Mutation; D011938:Rec A Recombinases / Q000378:metabolism*; D011995:Recombination, Genetic
D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases; D000251:Adenosine Triphosphatases