Role of walker motif A of RuvB protein in promoting branch migration of holliday junctions. Walker motif a mutations affect Atp binding, Atp hydrolyzing, and DNA binding activities of Ruvb.
Escherichia coli RuvB protein, an ATP-dependent hexameric DNA helicase, acts together with RuvA protein to promote branch migration of Holliday junctions during homologous recombination and recombinational repair. To elucidate the role of the Walker motif A of RuvB (GXGKT; X indicates a nonconserved residue) in ATP hydrolysis and branch migration activities, we constructed four ruvB mutant genes by site-directed mutagenesis, altering the highly conserved Lys(68) and Thr(69). K68R, K68A, and T69A mutants except T69S failed to complement UV-sensitive phenotype of the ruvB strain. These three mutant proteins, when overexpressed, made the wild-type strain UV-sensitive to varying degrees. K68R, K68A, and T69A were defective in ATP hydrolysis and branch migration activities in vitro. In the presence of Mg(2+), K68R showed markedly reduced affinity for ATP, while K68A and T69A showed only mild reduction. K68A and T69A could form hexamers in the presence of Mg(2+) and ATP, while K68R failed to form hexamers and existed instead as a higher oligomer, probably a dodecamer. In contrast to wild-type RuvB, K68R, K68A, and T69A by themselves were defective in DNA binding. However, RuvA could facilitate binding of K68A and T69A to DNA, whereas it could not promote binding of K68R to DNA. All of the three mutant RuvBs could physically interact with RuvA. These results indicate the direct involvement in ATP binding and ATP hydrolysis of the invariant Lys(68) and Thr(69) residues of Walker motif A of RuvB and suggest that these residues play key roles in interrelating these activities with the conformational change of RuvB, which is required for the branch migration activity.
Hishida
T
T
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Iwasaki
H
H
Yagi
T
T
Shinagawa
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004260:DNA Repair; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D011485:Protein Binding; D011995:Recombination, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; C061260:RuvB protein, Bacteria; D000255:Adenosine Triphosphate