Biochemical properties of RuvBD113N: a mutation in helicase motif II of the RuvB hexamer affects DNA binding and ATPase activities.
Many DNA helicases utilise the energy derived from nucleoside triphosphate hydrolysis to fuel their actions as molecular motors in a variety of biological processes. In association with RuvA, the E. coli RuvB protein (a hexameric ring helicase), promotes the branch migration of Holliday junctions during genetic recombination and DNA repair. To analyse the relationship between ATP-dependent DNA helicase activity and branch migration, a site-directed mutation was introduced into the helicase II motif of RuvB. Over-expression of RuvBD113N in wild-type E. coli resulted in a dominant negative UVs phenotype. The biochemical properties of RuvBD113N were examined and compared with wild-type RuvB in vitro. The single amino acid substitution resulted in major alterations to the biochemical activities of RuvB, such that RuvBD113N was defective in DNA binding and ATP hydrolysis, while retaining the ability to form hexameric rings and interact with RuvA. RuvBD113N formed heterohexamers with wild-type RuvB, and could inhibit RuvB function by affecting its ability to bind DNA. However, heterohexamers exhibited an ability to promote branch migration in vitro indicating that not all subunits of the ring need to be catalytically competent.
Mézard
C
C
Clare Hall Laboratories, Imperial Cancer Research Fund, South Mimms, Hertfordshire, EN6 3LD, UK.
Davies
A A
AA
Stasiak
A
A
West
S C
SC
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004265:DNA Helicases / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011995:Recombination, Genetic / Q000502:physiology*
D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061260:RuvB protein, Bacteria; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D004247:DNA; C071059:Holliday junction DNA helicase, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases