Site-directed mutagenesis of the RecA protein of Escherichia coli. Tyrosine 264 is required for efficient ATP hydrolysis and strand exchange but not for LexA repressor inactivation.
The role of Tyr264 in nucleotide binding and hydrolysis catalyzed by the RecA protein of Escherichia coli was investigated by constructing Gly, Ser, and Phe substitution mutations using oligonucleotide-directed mutagenesis. The corresponding mutant recA genes neither restored resistance to killing by ultraviolet irradiation nor increased homologous recombination in a recA strain. The purified RecA(Gly264) protein was unable to bind nucleotide, hydrolyze ATP, or form stable ternary complexes with adenosine 5'-O-thiotriphosphate and DNA although the mutant protein bound DNA normally in the absence of nucleotide. The RecA (Phe264) and RecA(Ser264) proteins hydrolyzed ATP poorly and the rates were reduced approximately 8- and 18-fold, respectively. Although capable of low levels of ATP hydrolysis, neither the RecA(Phe264) nor the RecA(Ser264) protein promoted DNA pairing or strand exchange reactions in vitro. Furthermore, these mutant RecA proteins were impaired in their ability to form salt-resistant ternary complexes with adenosine 5'-O-thiotriphosphate) and DNA as judged by filter binding. Nevertheless, nucleoprotein complexes formed with either RecA(Phe264) or RecA(Ser264) protein directed efficient cleavage of LexA repressor in vitro. These results demonstrate that Tyr264 is required for efficient ATP hydrolysis and for homologous pairing of DNA but does not participate in activating RecA protein for LexA repressor autodigestion.
Freitag
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NE
Laboratory of Biomedical and Environmental Sciences, UCLA School of Medicine 90024.
McEntee
K
K
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000302:isolation & purification*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism / Q000528:radiation effects; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D011485:Protein Binding; D011938:Rec A Recombinases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000302:isolation & purification*; D015183:Restriction Mapping; D012697:Serine Endopeptidases; D014443:Tyrosine; D014466:Ultraviolet Rays; D000099160:LexA Repressor Protein
D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D015345:Oligonucleotide Probes; D011938:Rec A Recombinases; D012097:Repressor Proteins; D012697:Serine Endopeptidases; D014443:Tyrosine; D000099160:LexA Repressor Protein