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Underpinning replication of protein-bound DNA by the accessory replicative helicase Rep

Brüning, Jan-Gert (0025) Underpinning replication of protein-bound DNA by the accessory replicative helicase Rep. PhD thesis, University of York.

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Accurate DNA replication must occur prior to every cell division. However, replication forks often stall at sites of DNA damage and protein-DNA complexes. If not removed, these blocks can threaten the viability of both daughter cells by preventing the completion of genome duplication or by targeting of blocked forks by recombination enzymes that can result in gross chromosomal rearrangements and genome instability. The importance of minimising fork blockage has resulted in cells evolving repair systems to remove lesions from DNA whilst accessory replicative helicases can underpin replication fork movement through hard-to-replicate sites including protein-DNA complexes. This thesis investigates the Escherichia coli accessory replicative helicase Rep. It is shown that efficient recruitment of Rep to the replisome via an interaction with the replicative helicase DnaB is dependent on the extreme Rep C terminus. This work also indicates that the DnaB C terminus is necessary for this interaction. Secondly, this work determines the function of the 2B subdomain, a conserved feature of Superfamily 1A (SF1A) helicases. Characterisation of a Rep mutant lacking this domain (RepΔ2B) showed greatly reduced levels of protein displacement from DNA, indicating a central role of the 2B subdomain in the removal of nucleoprotein blocks. Complementation of this mutation by a 2B subdomain of the homologous helicase UvrD supports the idea that the accessory replicative helicase function of Rep is dependent on a 2B subdomain. These data also demonstrate that the function of 2B subdomains is conserved among other SF1A helicases. Previous work had also shown that the 2B subdomain of SF1A helicases is flexible. Mutations in the hinge that connect the 2B subdomain to the rest of the helicase resulted in activation of DNA helicase activity and increased levels of nucleoprotein removal from single-stranded (ss) and double-stranded (ds) DNA. These data shed new light on how translocation along DNA is coupled to protein displacement during helicase catalysis, a conserved function of many helicases. A model is proposed where ATP hydrolysis is closely linked to conformational changes of the 2B subdomain of Rep, facilitating protein displacement by Rep.

Item Type: Thesis (PhD)
Academic Units: The University of York > Biology (York)
Identification Number/EthosID: uk.bl.ethos.638986
Depositing User: Mr Jan-Gert Brüning
Date Deposited: 11 Mar 2015 10:56
Last Modified: 24 Jul 2018 15:20
URI: http://etheses.whiterose.ac.uk/id/eprint/8220

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