Obstacles to DNA replication in Escherichia coli and the role of UvrD helicase in their resolution

UvrD is a multi-functional Escherichia coli helicase. It is widely involved in DNA repair, including its DNA unwinding role during mismatch repair and RNAP removal role during transcription coupled repair. UvrD has also been shown to colocalise with the replisome during DNA replication and has an important role in nucleoprotein block removal to clear the path for the replication fork.

The main aims of this project were to test UvrD ability to unwind DNA and displace nucleoprotein blocks and to test whether addition of the mismatch protein MutL could improve UvrD function. Our in vitro results showed that UvrD alone was capable of unwinding past a physiologically relevant block, Tus-terB, in ‘easy’ and ‘challenging’ conditions. The addition of MutL increased the ability of UvrD to unwind long double-stranded DNA substrates in multiple challenging conditions as well as against a physiologically unfamiliar nucleoprotein block (EcoRI E111G). Our results showed that UvrD activity appeared to be enhanced when tasked with unwinding past the Tus-terB block and presented better DNA unwinding alone rather than
with MutL. To understand more about the UvrD:MutL physical and functional interaction, we designed two UvrD mutants with large N-terminal deletions for UvrD:MutL interaction tests. Additionally, since UvrD:MutL complex formation causes the rotation of the UvrD 2B subdomain, we designed a UvrD∆2B mutant, to test for MutL interaction and determine how the 2B subdomain deletion affects UvrD function. We also developed a new in vitro biochemical assay designed to test for the displacement of Tus by the replisome during DNA replication termination. Our preliminary findings suggest that replication forks colliding with the non-permissive orientation of the Tus-ter block can displace Tus. Establishment of the Tus jumping assay will allow us to investigate the interaction of DNA replication forks with Tus-ter and to test whether other proteins, such as UvrD, are involved in replication termination.