ATP-dependent resolution of R-loops at the ColE1 replication origin by Escherichia coli RecG protein, a Holliday junction-specific helicase

Abstract

single strand DNA while RecG shows a 3Ј to 5Ј DNA Department of Molecular Microbiology, Research Institute for helicase activity (Tsaneva et al., 1993; Whitby et al., Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, 1994). Osaka 565, Japan In the RuvABC pathway, RuvC is a specific endonucle-1 Corresponding author ase that resolves Holliday junctions (Dunderdale et al., 1991;Iwasaki et al., 1991). RecG, which functions in The RecG protein of Escherichia coli is a DNA helicase the alternative pathway, has been suggested to promote that promotes branch migration of the Holliday juncjunction resolution by driving branch migration in the tions. We found that overproduction of RecG protein opposite direction from that promoted by RecA protein drastically decreased copy numbers of ColE1-type (Whitby et al., 1993;Whitby and Lloyd, 1995). These plasmids, which require R-loop formation between the authors proposed that RecG plays a role in preventing template DNA and a primer RNA transcript (RNA unproductive recombination by reversing the invasion of II) for the initiation of replication. RecG efficiently a 5Ј single-stranded DNA end into the homologous duplex inhibited in vitro ColE1 DNA synthesis in a reconstitu- . ted system containing RNA polymerase, RNase HI andThe recG and ruv genes play different roles in alternative DNA polymerase I. RecG promoted dissociation of mechanisms of stable DNA replication (SDR), which do RNA II from the R-loop in a manner that required not require the chromosomal replication origin (oriC), ATP hydrolysis. These results suggest that overDnaA initiator protein or concomitant protein synthesis produced RecG inhibits the initiation of replication (Asai and Kogoma, 1994a). Mutations in the ruv or recG by prematurely resolving the R-loops formed at the gene stimulate inducible SDR (iSDR), which is a part of replication origin region of these plasmids with its SOS responses, suggesting that this mode of SDR involves unique helicase activity. The possibility that RecG D-loops or Holliday junctions made by RecA (Asai and regulates the initiation of a unique mode of DNA Kogoma, 1994b). recG mutants, but not ruv mutants, replication, oriC-independent constitutive stable DNA stimulate constitutive SDR (cSDR), which was found replication, by its activity in resolving R-loops is disoriginally in RNase HI-defective (rnhA) mutants, and recG cussed.rnhA double mutants are inviable. These results suggested

Roles of the recG gene product of Escherichia coli in recombination repair: Effects of the .DELTA.recG mutation on cell division and chromosome partition.

“…RecA may bind to the displaced single-stranded DNA in the Rloops. Consistent with this idea, an rnhA mutant, lacking the RNase H1 activity and thus defective in resolving R-loops, chronically induces the SOS response, and RecG protein can also resolve the R-loops in an ATP-dependent manner (Kogoma et al, 1993;Vincent et al, 1996;Fukuoh et al, 1997).…”

RecG Protein and Single-Strand DNA Exonucleases Avoid Cell Lethality Associated With PriA Helicase Activity inEscherichia coli

“…These two proteins provide different ways of eliminating R-loops. RecG is a double-stranded DNA (dsDNA) translocase and dissociates the RNA from the structure by catalyzing branch migration whereas RNase HI digests the RNA from the RNA:DNA hybrid (Hong et al 1995;Vincent et al 1996;Fukuoh et al 1997;McGlynn et al 1997;Singleton et al 2001). Strains lacking both proteins are inviable, indicating that excessive levels of SDR may be harmful (Hong et al 1995).…”

Situational Repair of Replication Forks

“…In addition to its action on DNA substrates, RecG removes R-loops in vivo and in vitro (37). RecG-mediated R-loop removal renders the recG gene essential for the viability of RNase H mutants (rnh), and recG inactivation triggers the use of R-loops as alternative replication initiation points, a phenomenon termed constitutive stable DNA replication (38).…”