Journal of Molecular Biology

Accessory helicases UvrD and Rep in E. coli act by distinctly differently mechanisms.

UvrD functions at a tetramer at the replication fork.

The interaction of UvrD at the fork is not mediated by sequence specifity.

Frequent blocks to replication influence UvrD interaction with the fork.

The amount of UvrD at the fork is dependent upon its block removal function.

DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help remove these blocks and aid the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD functions between its activities in DNA repair and role as an accessory helicase remain unclear in live cells. By integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into tetrameric assemblies and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its colocation with forks is likely due to the very high frequency of replication blocks composed of DNA-bound proteins, including RNA polymerase and factors involved in repairing 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 UvrD’s function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication.

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Super-resolved single-molecule light microscopy indicates fork-UvrD colocation. (A). Slimfield microscope schematic and micrographs of mGFP-UvrD, DnaQ-mCherry. Cell membrane and DnaQ/UvrD foci indicated as white dashed and non-dashed lines respectively. (B) and (C) Histogram showing the number of DnaQ and UvrD foci detected per cell respectively, SD errors. N = 45 cells.

UvrD helicase

genome stability

replication

polymerase

fluorescence microscopy

single molecule

© 2023 The Authors. Published by Elsevier Ltd.