BIOCHEMICAL-PROPERTIES OF RUVB(D113N) - 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 triphosp hate hydrolysis to fuel their actions as molecular motors in a variety of biological processes. In association with RuvA, the E. coli RuvB p rotein (a hexameric ring helicase), promotes the branch migration of H olliday junctions during genetic recombination and DNA repair. To anal yse the relationship between ATP-dependent DNA helicase activity and b ranch migration, a site-directed mutation was introduced into the heli case II motif of RuvB. Over-expression of RuvB(D113N) in wild-type E. coli resulted in a dominant negative UVs phenotype. The biochemical pr operties of RuvB(D113N) were examined and compared with wild-type RuvB in vitro. The single amino acid substitution resulted in major altera tions to the biochemical activities of RuvB, such that RuvB(D113N) was defective in DNA binding and ATP hydrolysis, while retaining the abil ity to form hexameric rings and interact with RuvA. RuvB(D113N) formed heterohexamers with wild-type RuvB, and could inhibit RuvB function b y affecting its ability to bind DNA. However, heterohexamers exhibited an ability to promote branch migration in vitro indicating that not a ll subunits of the ring need to be catalytically competent. (C) 1997 A cademic Press Limited.