PT  - JOURNAL ARTICLE
AU  - Wollman, Adam J. M
AU  - Syeda, Aisha H.
AU  - Leech, Andrew
AU  - Guy, Colin
AU  - McGlynn, Peter
AU  - Hawkins, Michelle
AU  - Leake, Mark C.
TI  - Tetrameric UvrD helicase is located at the <em>E. coli</em> replisome due to frequent replication blocks
AID  - 10.1101/2021.02.22.432310
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.02.22.432310
4099  - http://biorxiv.org/content/early/2021/02/22/2021.02.22.432310.short
4100  - http://biorxiv.org/content/early/2021/02/22/2021.02.22.432310.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.