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Tetrameric UvrD helicase is located at the E. coli replisome due to frequent replication blocks

Adam J. M Wollman, Aisha H. Syeda, Andrew Leech, Colin Guy, Peter McGlynn, Michelle Hawkins, View ORCID ProfileMark C. Leake
doi: https://doi.org/10.1101/2021.02.22.432310
Adam J. M Wollman
1Department of Physics, University of York, York YO10 5DD, United Kingdom
2Department of Biology, University of York, York YO10 5DD, United Kingdom
3Biosciences Institute, Newcastle University, NE1 7RU, United Kingdom
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Aisha H. Syeda
1Department of Physics, University of York, York YO10 5DD, United Kingdom
2Department of Biology, University of York, York YO10 5DD, United Kingdom
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Andrew Leech
4Bioscience Technology Facility, Department of Biology, University of York, York YO10 5DD, United Kingdom
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Colin Guy
5Covance Laboratories Ltd., Otley Road, Harrogate, HG3 1PY, United Kingdom
6School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom
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Peter McGlynn
2Department of Biology, University of York, York YO10 5DD, United Kingdom
6School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, United Kingdom
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Michelle Hawkins
2Department of Biology, University of York, York YO10 5DD, United Kingdom
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Mark C. Leake
1Department of Physics, University of York, York YO10 5DD, United Kingdom
2Department of Biology, University of York, York YO10 5DD, United Kingdom
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  • ORCID record for Mark C. Leake
  • For correspondence: mark.leake@york.ac.uk
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ABSTRACT

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 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

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
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Posted February 22, 2021.
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Tetrameric UvrD helicase is located at the E. coli replisome due to frequent replication blocks
Adam J. M Wollman, Aisha H. Syeda, Andrew Leech, Colin Guy, Peter McGlynn, Michelle Hawkins, Mark C. Leake
bioRxiv 2021.02.22.432310; doi: https://doi.org/10.1101/2021.02.22.432310
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Tetrameric UvrD helicase is located at the E. coli replisome due to frequent replication blocks
Adam J. M Wollman, Aisha H. Syeda, Andrew Leech, Colin Guy, Peter McGlynn, Michelle Hawkins, Mark C. Leake
bioRxiv 2021.02.22.432310; doi: https://doi.org/10.1101/2021.02.22.432310

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