UCL Discovery

Abstract

The RuvA and RuvB proteins of Escherichia coil 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.