Abstract
Playing important roles in rolling circle DNA replication, homologous recombination and DNA repair, PcrA belongs to the well-established UvrD-like family of helicases. PcrA’s involvement in many DNA transactions is directed by interactions with several partner proteins, including RNA polymerase (RNAP). Recent structural data uncovered interactions between the core of PcrA and areas surrounding the DNA and RNA exit channels of RNAP. Additional contacts between PcrA’s C-terminal domain and RNAP’s SI1 domain identified a putative helicase interaction motif (HIM), also found in several other PcrA interactors. Furthermore, biochemical assays implicated PcrA in DNA:RNA hybrid unwinding and R-loop homeostasis, but the mechanism behind this remains unknown.A high resolution structure of the PcrA:RNAP complex is now required to determine its precise contacts and to assist in establishing the mechanism by which PcrA resolves R-loops. In this thesis, several methods for formation of a stable PcrA:RNAP complex for downstream imaging applications are explored. We find that individually combining native B.subtilis RNAP with recombinant B.subtilis PcrA in the presence of an artificial DNA:RNA scaffold yields a stable complex but that other approaches, including co-expression of interaction partners and the use of recombinant RNAP, fail. Moreover, EMSAs showed that multiple shifted species were generated upon addition of PcrA to the RNAP:scaffold complex, revealing an unexpected complexity in the species formed that requires further investigation.
As an alternative and complementary approach to understanding the interactions of PcrA with partner proteins, we use AlphaFold-Multimer to model PcrA complexes with partners containing a HIM. High confidence contacts were predicted between PcrA’s CTD and both the SI1 domain of RNAP and a region of YxaL containing the HIM, yielding testable models for the complexes formed. A lower confidence interaction was also predicted between PcrA’s 2B domain and RNAP’s β-subunit, which is consistent with structural data, but requires experimental validation.
| Date of Award | 20 Jun 2023 |
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| Original language | English |
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| Supervisor | Mark S Dillingham (Supervisor), Alan C M Cheung (Supervisor) & Nigel J Savery (Supervisor) |