SUMMARY
DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help replication machinery overcome blocks by removing incoming nucleoprotein complexes or aiding the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD between its multiple activities in DNA repair and its role as an accessory helicase remains unclear in live cells. Here, by integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into a tetramer and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its recruitment to forks is likely mediated by the very high frequency of replication blocks due to DNA bound proteins, including RNA polymerase and DNA damage. Deleting rep and DNA repair factor genes mutS and uvrA, and inhibiting transcription through RNA polymerase mutation and antibiotic inhibition, indicates that the level of UvrD at the fork is dependent on its function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via distinctly different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors
Minor rewording to abstract; localisation microscopy graphical markers added to detected fluorescent foci images
