Cell proliferation requires high fidelity DNA replication of all genetic material. However, DNA replication occurs on template DNA coated in nucleoprotein complexes. The inability of the replisome, a multiprotein replication machine, to overcome nucleoprotein obstacles has been determined as a major source of genetic instability in many organisms. The bacterial model organism, Escherichia coli (E. coli), contains the accessory helicase, Rep, that can remove these nucleoprotein barriers from the path of the replication fork, rescuing stalled replication. The interactions between Rep and the replisome that contribute to this activity remain unknown.
To investigate if Rep can rescue stalled replication forks in vitro we developed a model nucleoprotein complex using the nuclease dead CRISPR/Cas9 (dCas9) protein. Using bulk biochemical replication assays and single-molecule Total Internal Reflection Fluorescence (smTIRF), we show that replication is site-specifically stalled by high-stability dCas9 complexes, evident by the absence of extended DNA products. Further, these roadblocks are displaced by Rep, allowing replication to continue, which is indicated by the extension of previously blocked DNA products. Observations of fluorescently labelled Rep in smTIRF assays suggest that in the absence of roadblocks, Rep continuously associates with the replisome in a dynamic manner. However, in the presence of dCas9 roadblocks, an increased recruitment of Rep to the sites of stalled replication result in the displacement of the roadblock and the resumption of replication. These observations suggest that Rep continuously surveys the state of the replisome and act when required to remove high-stability nucleoprotein roadblocks from the path of the replication fork. Additionally, use of the dCas9 roadblock provides insight into how the replisome may overcome commonly encountered nucleoprotein complexes in vivo, for example, RNA polymerase.