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Title: Bacterial DNA replication initiation : structural and functional analysisof the master initiator DnaA
Authors: Stevens, Daniel
Issue Date: 2020
Publisher: Newcastle University
Abstract: The essential process of DNA replication begins with initiator proteins binding to origins of replication and triggering DNA synthesis. The highly conserved bacterial master initiator protein, DnaA, performs several key activities at the bacterial origin (oriC) to initiate replication. DnaA binds specifically to oriC and assembles into a filament that engages and stretches a single DNA strand to induce duplex unwinding. Subsequently, DnaA recruits a loading complex that deposits the replicative helicases around single DNA strands. In this thesis I have investigated the molecular mechanisms underpinning some of the essential activities of DnaA in the model organism Bacillus subtilis. Using a chimeric DnaA system I was able to identify several activities required for origin unwinding by DnaA bound to a specific DnaA-box located upstream of the site of unwinding. This result suggested that the protein binding here is directly involved in unwinding the DNA duplex, and the likely role of the upstream region is to increase the local DnaA concentration at the site of unwinding.To unwind oriC, DnaA engages and stretches a specific DNA strand with a recently identified repeating tri-nucleotide motif, termed the DnaA-tri os, providing the specific sequence. Utilising an inducible heterologous replication initiation system I determined which DnaA residues from a region implicated in ssDNA binding were essential in vivo. Using recombinant DnaA protein variants, two isoleucine residues were determined to be required for forming filaments on ssDNA and unwinding the DNA duplex in vitro. Further work is required to determine if these residues are required for the specific interaction with DnaA-trios or more generally for DNA binding/unwinding. A range of essential residues required for the interaction between DnaA and thefirmicute specific initiation accessory protein DnaD, the first step in helicase recruitment, were identified. The DnaA residues overlap with a binding site for the developmental regulator, SirA, a developmentally expressed inhibitor of DNA replication initiation. This suggested that SirA functions by blocking the interaction between DnaA and DnaD, preventing helicase loading. I found that SirA inhibits the interaction of DnaA with DnaD, providing a molecular mechanism for this SirA activity and revealing, for the first time, an endogenous system for regulating helicase recruitment in bacteria
Description: PhD Thesis
Appears in Collections:Institute for Cell and Molecular Biosciences

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