Analysis of the DNA binding site of Escherichia coli RecA protein.
To investigate the DNA binding site of RecA protein, we constructed 15 recA mutants having alterations in the regions homologous to the other ssDNA binding proteins. The in vivo analyses showed that the mutational change at Arg243, Lys248, Tyr264, or simultaneously at Lys6 and Lys19, or Lys6 and Lys23 caused severe defects in the recA functions, while other mutational changes did not. Purified RecA-K6A-K23A (Lys6 and Lys23 changed to Ala and Ala, respectively) protein was indistinguishable from the wild-type RecA protein in its binding to DNA. However, the RecA-R243A (Arg243 changed to Ala) and RecA-Y264A (Tyr264 changed to Ala) proteins were defective in binding to both ss- and ds-DNA. In self-oligomerization property, RecA-R243A was proficient but RecA-Y264A was deficient, suggesting that the RecA-R243A protein had a defect in DNA binding site and the RecA-Y264A protein was defective in its interaction with the adjacent RecA molecule. The region of residues 243-257 including the Arg243 is highly homologous to the DNA binding motif in the ssDNA binding proteins, while the eukaryotic RecA homologues have a similar structure at the amino-terminal side proximal to the nucleotide binding core. The region of residues 243-257 would be a part of the DNA binding site. The other parts of this site would be the Tyr103 and the region of residues 178-183, which were cross-linked to ssDNA. These three regions lie in a line in the crystal structure.
Morimatsu
K
K
Department of Molecular Protozoology, Osaka University, Japan.
Horii
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011938:Rec A Recombinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004269:DNA, Bacterial; D011938:Rec A Recombinases