Proteins that bind DNA are the cause of the majority of impediments to replication fork progression and can lead to subsequent collapse of the replication fork. Failure to deal with fork collapse efficiently leads to mutation or cell death. Several models have been proposed for how a cell processes a stalled or collapsed replication fork; eukaryotes and bacteria are not dissimilar in terms of the general pathways undertaken to deal with these events. This study shows that replication fork regression, the combination of replication fork reversal leading to formation of a Holliday Junction along with exonuclease digestion, is the preferred pathway for dealing with a collapsed fork in Escherichia coli. Direct endo-nuclease activity at the replication fork was not observed. The protein that had the greatest effect on these fork processing events was the RecQ helicase, while RecG and RuvABC, which have previously been implicated in this process, were found to play a lesser role. Eukaryotic RecQ homologues, BLM and WRN, have also been implicated in processing events following replication fork collapse and may reflect a conserved mechanism. Finally, the SOS response was not induced by the protein-DNA roadblock under these conditions, so did not affect fork processing.

Obstacles present on DNA, including tightly-bound proteins and various lesions, can severely inhibit the progression of the cell's replication machinery. The stalling of a replisome can lead to its dissociation from the chromosome, either in part or its entirety, leading to the collapse of the replication fork. The recovery from this collapse is a necessity for the cell to accurately complete chromosomal duplication and subsequently divide. Therefore, when the collapse occurs, the cell has evolved diverse mechanisms that take place to restore the DNA fork and allow replication to be completed with high fidelity. Previously, these replication repair pathways in bacteria have been studied using UV damage, which has the disadvantage of not being localized to a known site. This manuscript describes a system utilizing a Fluorescence Repressor Operator System (FROS) to create a site-specific protein block that can induce the stalling and collapse of replication forks in Escherichia coli. Protocols detail how the status of replication can be visualized in single living cells using fluorescence microscopy and DNA replication intermediates can be analyzed by 2-dimensional agarose gel electrophoresis. Temperature sensitive mutants of replisome components (e.g. DnaBts) can be incorporated into the system to induce a synchronous collapse of the replication forks. Furthermore, the roles of the recombination proteins and helicases that are involved in these processes can be studied using genetic knockouts within this system.

No abstract

There 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 in Escherichia 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 of Escherichia 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.

No abstract