Replication termination

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The Escherichia coli genome contains a replication fork trap which is mediated by unidirectional Tus-ter barriers. The fusing of replication forks to complete genome duplication is a key process in all organisms and is crucial for genome stability in E. coli. Our group uses biochemical reconstitution to investigate what happens when replication forks fuse. We are beginning to broaden these studies using structural biology techniques. This will enable us to characterise the structure of replisome-Tus complexes and gain a mechanistic understanding of the final stage of DNA replication.

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Replication-transcription conflicts

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When DNA replication is impeded by blocks it can lead to genome instability or cell death. Barriers to DNA replication are a problem for all life but are particularly important in bacteria because their genomes are replicated from a single bidirectional replication origin. This means that replisomes from another nearby origin cannot rescue replisomes that are stalled at blocks. Replication and transcription of DNA occur simultaneously in E. coli so RNA polymerase is the most common obstacle encountered by the replisome. Accessory replicative helicases promote fork movement through nucleoprotein barriers and have been identified in prokaryotes and eukaryotes. Rep is the primary accessory helicase in E. coli. UvrD is a Rep homologue that can partially compensate for the absence of Rep. Both helicases reduce replisome pausing in vivo and in vitro and at least one is required for E. coli survival. We are exploring suppressors of the Rep-UvrD synthetic lethality in order to further understand the balance between replication and transcription.

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UvrD function

UvrD is a versatile superfamily 1 E. coli helicase involved in a wide range of processes, including replication-transcription conflict resolution, mismatch repair, nucleotide excision repair, and DNA-bound protein block removals. UvrD is formed of four subdomains (1A, 1B, 2A, 2B) and requires ATP to function. Research has also demonstrated UvrD unwinding DNA both as a monomer and a dimer, with the in vivo form being widely debated by researchers. 

 

We are interested in the ability of UvrD to remove DNA-bound proteins, such as RNAP and Tus, and are currently investigating the role of its N-terminal and the 2B subdomain in protein removal. Ordavayev et al., 2018 and 2019 described the role of the 2B subdomain in DNA unwinding and the connection between MutL binding to UvrD helicase and the rotation of the 2B subdomain. Another superfamily 1 helicase, Rep, has been shown to be unable to remove DNA-bound proteins with its 2B subdomain was removed, and due to the structural and functional similarities between the two helicases, we are interested in finding out whether the removal of the UvrD 2B subdomain will generate a similar effect.