Mutagenesis of the P-loop motif in the ATP binding site of the RecA protein from Escherichia coli.
Using a combinatorial cassette mutagenesis procedure we have introduced a number of mutations into 10 codons that define the P-loop motif within the ATP binding site of the Escherichia coli RecA protein. The recombinational proficiency of the recA mutants was determined using three genetic assays: survival in the presence of 4-nitroquinoline-1-oxide, survival following UV irradiation and the ability to support plaque formation by a red-gam-Chi+ lambda phage. While no amino acid substitutions were allowed at the four residues that define the P-loop consensus sequence, a variety of changes at the other positions in this region were observed that allowed full or partial RecA function. This occurred despite the fact that these residues are very highly conserved among 22 eubacterial RecA proteins, and represent the most conserved stretch of 10 contiguous residues in the entire RecA sequence. Our results show that these residues display marked differences in the ability to support mutations. The mutability of each of these 10 residues is discussed in terms of possible functional and/or structural roles.
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655.
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D015112:4-Nitroquinoline-1-oxide / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003062:Codon / Q000235:genetics; D016384:Consensus Sequence; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics* / Q000528:radiation effects; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D011938:Rec A Recombinases / Q000032:analysis / Q000235:genetics* / Q000378:metabolism*; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays; D010948:Viral Plaque Assay
D003062:Codon; D015112:4-Nitroquinoline-1-oxide; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases