  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 activities of UvrD in vivo remain unclear. Here, by integrating
biochemical analysis and super-resolved single-molecule fluorescence
microscopy, we discovered 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. By deleting rep and DNA repair factors
mutS and uvrA, perturbing transcription by mutating RNA polymerase, and
antibiotic inhibition; we show that the presence of UvrD at the fork is
dependent on its activity. This is likely mediated by the very high frequency
of replication blocks due to DNA bound proteins, including RNA polymerase, and
DNA damage. UvrD is recruited to sites of nucleoprotein blocks via distinctly
different mechanisms to Rep and therefore plays a more important and
complementary role than previously realised in ensuring successful DNA
replication.

Biomolecules