RuvA and RuvB proteins of Escherichia coli exhibit DNA helicase activity in vitro.

Abstract

The SOS-inducible ruvA and ruvB gene products ofEscherichia coli are required for normal levels ofgenetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5' -b 3' polarity. The reaction is ATPdependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.DNA helicases play essential roles in DNA replication, repair, and recombination (for review see ref. 1). In bacteria, helicases such as Rep, DnaB, and PriA (n') act at the replication fork, where they unwind DNA during replication (2). Unwinding occurs with a defined polarity and is driven at the expense of nucleoside triphosphate hydrolysis. In DNA repair, the UvrA and UvrB proteins, part of the UvrABC excision nuclease complex, exhibit helicase activity during the recognition of DNA lesions (3), while UvrD (DNA helicase II) is involved in the disassembly of post-incision complexes (4).During genetic recombination in Escherichia coli, the formation of recombinant DNA molecules occurs via a series of well-defined, yet overlapping steps, several of which involve the action of DNA helicases. For example, RecBCD enzyme unwinds duplex DNA leading to the initiation of recombination by RecA protein (5, 6). A similar role is likely to be played by the RecQ helicase (7). In early studies with RecA protein, it was thought that the mechanisms ofhomologous pairing and strand exchange might involve strand separation. However, this was not the case (8) and current work indicates the formation of multistranded DNA helices within the RecA filament (9-14). Nevertheless, the concept that subsequent branch migration of a Holliday junction and the formation of extensive lengths of heteroduplex DNA might be catalyzed by a helicase-like activity remains attractive.In recent studies we focused our attention on the proteins encoded by the ruv locus of the E. coli chromosome. The RuvA and RuvB proteins interact with each other and catalyze reactions that are important for genetic recombination and the recombinational repair of DNA damage. Early genetic studies showed that ruvA and ruvB mutants had similar phenotypes characteristic of a defect in a late step of recombination, such as the processing of recombination intermediates (15-17). Biochemical studies provided support for this notion by demonstrating that RuvA and RuvB together promote the branch migration of Holliday junctions in vitro, leading to the formation of heteroduplex DNA (...

Saccharomyces cerevisiae Mer3 Is a DNA Helicase Involved in Meiotic Crossing Over

“…Similar strand displacement reactions have been shown to be carried out by comparable amounts of other helicases thought to function in recombination, such as E. coli RuvAB and RecQ, yeast Sgs1 and Srs2, and human BLM (5,30,35,46,59,60). In S. cerevisiae, there are a number of genes encoding predicted DNA helicases that have been shown to be involved in DNA recombination, including SGS1, SRS2, RAD54, TID1/RDH54, and MER3 (14,33,41,49,53,64,65).…”

Characterization of the biochemical properties of the human Upf1 gene product that is involved in nonsense-mediated mRNA decay

“…Density gradient centrifugation analysis was performed to correlate the ATPase activity of the purified fraction with that of the HUpf1 protein+ The yeast Upf1p sediments as a monomer in solution (Czaplinski et al+, 1995)+ Many helicases, however, can be found that function in multimeric states (Reha-Krantz & Hurwitz, 1978;Finger & Richardson, 1982;Dykstra et al+, 1984;Bernstein & Richardson, 1988;Mastrangelo et al+, 1989;Rozen et al+, 1990;Chao & Lohman, 1991;Seo et al+, 1991;Li et al+, 1992;Tsaneva et al+, 1993)+ Therefore, to determine whether HUpf1 functions as a monomer, purified HUpf1 was loaded on a 15-35% glycerol gradient+ In parallel, proteins of known molecular weights were loaded on identical gradients+ The samples were centrifuged, gradients were fractionated and aliquots from the fractions were subjected to either SDS-PAGE followed by immunoblotting or assayed for their ATPase activity+…”

Assembly of the Escherichia coli RuvABC resolvasome directs the orientation of Holliday junction resolution

“…RuvA also targets RuvB to the junction (Parsons and West 1993). RuvB is a hexameric ring helicase that acts as the motor of branch migration (Tsaneva et al 1993;Stasiak et al 1994). Electron microscopic visualization of the RuvAB-Holliday junction complex revealed that the junction lies sandwiched between two RuvA tetramers and that branch migration is driven by two oppositely oriented RuvB rings that bind to opposing arms (Parsons et al 1995;Yu et al 1997).…”