Interaction of Escherichia coli RuvA and RuvB proteins with synthetic Holliday junctions.

Abstract

The RuvA, RuvB, and RuvC proteins of Eseherichia coli are required for the recombinational repair of ultraviolet light-or chemical-induced DNA damage. In vitro, RuvC protein interacts with Holliday junctions in DNA and promotes their resolution by endonucleolytic cleavage. In this paper, we investigate the interaction of RuvA and RuvB proteins with model Holliday junctions. Using band-shift assays, we show that RuvA binds synthetic Holliday structures to form specific protein-DNA complexes. Moreover, in the presence of ATP, the RuvA and RuvB proteins act in concert to promote dissociation of the synthetic Holliday structures. The dissociation reaction requires both RuvA and RuvB and a nucleotide cofactor (ATP or dATP) and is rapid (40% of DNA molecules dissociate within 1 min). The reaction does not occur when ATP is replaced by either ADP or the nonhydrolyzable analog of ATP, adenosine 5'-[v-thioltriphosphate. We suggest that the RuvA and RuvB proteins play a specific role in the branch migration of Holliday junctions during postreplication repair of DNA damage in E. coil.Mutations in the ruvA, ruvB, or ruvC genes of Escherichia coli give rise to mutants that are phenotypically very similar.

The directionality of RuvAB‐mediated branch migration: in vitro studies with three‐armed junctions

“…In previous studies, RuvA and RuvB were shown to Directionality of RuvAB-mediated branch migration promote branch migration of Holliday junctions in vitro (Iwasaki et al 1992;Müller et al 1993;Parsons et al 1992;Parsons & West 1993;Tsaneva et al 1992b). The structure of the RuvAB-Holliday junction complex was analysed by electron microscopy and DNase I protection, which showed that the complex is tripartite, with RuvA sandwiched between two hexameric rings of RuvB (Hiom & West 1995;Parsons et al 1995a).…”

Mutational analysis on structure–function relationship of a Holliday junction specific endonuclease RuvC

“…The concerted actions of RuvA, RuvB and RuvC proteins complete the homologous recombination process by resolving the Holliday junctions into two recombinant duplex DNA molecules West 1997). RuvA and RuvB form a complex that promotes branch migration (Iwasaki et al 1992;Parsons et al 1992;Tsaneva et al 1992). In this reaction, RuvA specifically binds the junction and targets RuvB to it (Iwasaki et al 1992;Parsons et al 1992).…”

“…RuvA and RuvB form a complex that promotes branch migration (Iwasaki et al 1992;Parsons et al 1992;Tsaneva et al 1992). In this reaction, RuvA specifically binds the junction and targets RuvB to it (Iwasaki et al 1992;Parsons et al 1992). RuvB protein forms a hexameric ring (Stasiak et al 1994) and possesses an ATP-dependent helicase activity that provides the driving force for branch migration (Tsaneva et al 1993).…”

Dissociation of RecA filaments from duplex DNA by the RuvA and RuvB DNA repair proteins.

“…Biochemical studies have shown that the RuvA and RuvB proteins interact with Holliday junctions to promote their movement along DNA, by a process known as branch migration (6)(7)(8). The RuvC protein resolves Holliday junctions by endonucleolytic cleavage (9,10).…”

“…RuvA (22 kDa) and RuvB (37 kDa) each play a defined role in branch migration, with RuvA targeting RuvB to the Holliday junction (11,12). The selective binding of RuvA to junctions (7,11), the stimulation of RuvB's ATPase by RuvA/DNA (8), and observations which show that saturating concentrations of RuvB alone can promote branch migration (13) suggest that RuvB is the motor that drives strand exchange during branch migration, with RuvA playing an ancillary role (12). Further biochemical studies of RuvAB have revealed a DNA helicase activity that unwinds DNA with a 5'-to-3' polarity (14).…”