Purification and biochemical characterization of Escherichia coli RecA proteins mutated in the putative DNA binding site.
Escherichia coli RecA protein plays a central role both in DNA repair and in recombination. We report biochemical properties of three new RecA proteins mutated at positions 199 (RecA694), 207 (RecA659), and 211 (RecA611) in the putative DNA binding site. RecA694 had a wild-type phenotype, whereas RecA611 and RecA659 were deficient in promoting both the self-cleavage of LexA repressor and the DNA-strand exchange reaction. In order to determine the origin of this inhibition, we examined the capacity of wild-type and mutant proteins to bind to single-stranded DNA (with and without single-stranded binding protein, SSB), double-stranded DNA, and ATP. DNA strand exchange defects were correlated with the inability of mutant proteins to displace SSB from DNA. For the recA659 mutation this inhibition was reversed by equimolar wild-type protein. In contrast, mixtures of either wild-type/RecA659 or wild-type/RecA611 proteins remained deficient in LexA cleavage, suggesting that the dominant negative phenotype of the mutant proteins may be a consequence of the formation heterologous RecA complexes. Various mutations in the putative DNA binding site of RecA protein altered ATP binding, ATPase activity, displacement of SSB from single-stranded DNA, and protein-protein interactions. These results are consistent with the hypothesis that DNA binding to this site of RecA relays allosteric effects to several functional domains throughout the protein.
Cazaux
C
C
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, Centre National de la Recherche Scientifique UPR 8221, Toulouse, France.
Larminat
F
F
Villani
G
G
Johnson
N P
NP
Schnarr
M
M
Defais
M
M
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011938:Rec A Recombinases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D001426:Bacterial Proteins; D017931:DNA Primers; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011938:Rec A Recombinases; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D000099160:LexA Repressor Protein; C037693:single stranded DNA dependent ATPase