title: Targeted versus non-targeted DNA helicase activity of the RuvA and RuvB proteins of Escherichia coli.
abstract: The RuvA and RuvB proteins of Escherichia coli promote the branch migration of Holliday junctions in vitro. To understand the relationship between branch migration and the intrinsic 5'-->3' DNA helicase activity of RuvAB, the requirements and substrate specificity of the helicase reaction have been studied in more detail. We find that RuvAB-mediated DNA unwinding and branch migration reactions show similar requirements for Mg2+ and ATP and are inhibited to a similar extent by ADP and ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)). The helicase activity, measured by the dissociation of a short fragment from circular single-stranded DNA, requires both RuvA and RuvB and is stimulated by subsaturating concentrations of single-strand binding protein (SSB). In contrast, saturating concentrations of SSB are inhibitory. Using substrates that contain a DNA junction, which permits the specific binding of RuvA, we find that the RuvA and RuvB proteins promote two types of helicase reactions: nonspecific reactions, which are sensitive to inhibition by stoichiometric amounts of SSB, and junction-targeted reactions, which are not inhibited by SSB. Using three-armed structures, we observe that junction-targeted reactions display a polarity and result in asymmetric product formation. Junction-specific binding and the subsequent initiation of DNA unwinding are likely to represent early steps in the process of branch migration.
authors.affiliation: Imperial Cancer Research Fund, Clare Hall Laboratories, South Mimms, Herts., United Kingdom.
authors.forename: I R
authors.initials: IR
authors.lastname: Tsaneva
authors.forename: S C
authors.initials: SC
authors.lastname: West
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C061260:RuvB protein, Bacteria; D004247:DNA; C071059:Holliday junction DNA helicase, E coli; D000251:Adenosine Triphosphatases; D004265:DNA Helicases