Replication fork collapse at a protein-DNA roadblock leads to fork reversal, promoted by the RecQ helicase

Abstract

AbstractThere are numerous impediments that DNA replication can encounter while copying a genome, including the many proteins that bind DNA. Collapse of the replication fork at a protein roadblock must be dealt with to enable replication to eventually restart; failure to do so efficiently leads to mutation or cell death. Several prospective models have been proposed that process a stalled or collapsed replication fork. This study shows that replication fork reversal (RFR) is the preferred pathway for dealing with a collapsed fork inEscherichia coli, along with exonuclease activity that digests the two nascent DNA strands. RFR moves the Y-shaped replication fork DNA away from the site of the blockage and generates a four-way DNA structure, the Holliday junction (HJ). Direct endo-nuclease activity at the replication fork is either slow or does not occur. The protein that had the greatest effect on HJ processing/RFR was found to be the RecQ helicase. RecG and RuvABC both played a lesser role, but did affect the HJ produced: mutations in these known HJ processing enzymes produced longer-lasting HJ intermediates, and delayed replication restart. The SOS response is not induced by the protein-DNA roadblock under these conditions and so does not affect fork processing.Author SummaryTo transfer genetic material to progeny, a cell must replicate its DNA accurately and completely. If a cell does not respond appropriately to inhibitors of the DNA replication process, genetic mutation and cell death will occur. Previous works have shown that protein-DNA complexes are the greatest source of replication fork stalling and collapse in bacteria. This work examines how the cell deals with replication fork collapse at a persistent protein blockage, at a specific locus on the chromosome ofEscherichia coli. Cells were found to process the DNA at the replication fork, moving the branch point away from the site of blockage by replication fork reversal and exonuclease activity. Our data indicate that it is the RecQ helicase that has the main controlling role in this process, and not the proteins RecG and RuvABC, as currently understood. RecQ homologs have been shown to be involved in replication fork processing in eukaryotes and their mutation predisposes humans to genome instability and cancer. Our findings suggest that RecQ proteins could play more important role in replication fork reversal than previously understood, and that this role could be conserved across domains.