Functional characterization of residues in the P-loop motif of the RecA protein ATP binding site.
We have introduced a large number of single amino acid substitutions at six positions that lie within the P-loop motif of the ATP binding site in the Escherichia coli RecA protein. The activity of each recA mutant was determined using genetic assays which assess the catalytic proficiency of the RecA protein for both homologous genetic recombination and recombinational repair of damaged DNA. The six residues displayed unique patterns of allowed versus non-allowed substitutions that define the functional and structural constraints at each position. Our results show that while the restricted mutability of Gly66 and Ser70 conform to expectations based on their positions in the RecA crystal structure, strict constraints are in effect at positions 68 and 69 that are not apparent in the RecA structure but would be compatible with the proximity of these side-chains to bound DNA. Thr74 shows a rather unexpected pattern of allowed substitutions that may reflect two distinct structural solutions to optimal stabilization of bound nucleotide. In addition, specific substitutions at Pro67 result in mutant RecA proteins that appear to discriminate between homologous genetic recombination and recombinational DNA repair.
Konola
J T
JT
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester 01655.
Logan
K M
KM
Knight
K L
KL
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D003062:Codon; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010641:Phenotype; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic / Q000502:physiology; D013329:Structure-Activity Relationship
D003062:Codon; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases