Molecular mimicry in the loading of a bacterial recombinase by a phage mediator

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The genetic material in our cells is subject to frequent threats to its integrity, especially while undergoing replication to produce new cell copies. Multiple repair and restoration pathways have evolved to ensure faithful transfer of information from generation to generation. Defects in these processes lead to cancer in higher organisms and significantly impair survival in simpler forms of life such as bacteria. Our group studies the recombinational repair pathway, which utilises the unique pattern of nucleotides stored in the DNA double helix as a template for repair of a damaged partner chromosome. In all organisms, the same enzyme (called RecA or Rad51) is utilised to exchange a single strand from one DNA helix to another as the first step in damage restoration. Both bacterial RecA and human Rad51 strand exchange recombinases polymerise on single-strands of DNA to form long filament-like structures. Unfortunately loading of these recombination enzymes is hindered by single-stranded DNA binding proteins that protect the template from further damage. To overcome this potentially serious predicament, cells contain specialized helper proteins that assist RecA and Rad51 in gaining access to the DNA strands. Despite considerable research effort, the detailed mechanism of RecA and Rad51 facilitated assembly remains unclear. We have discovered a new helper activity, from a virus infecting bacteria, with the capacity to hijack the bacterial RecA protein to promote repair of its own genetic material. This protein, Orf, has some interesting features in common with BRCA2, which helps load the Rad51 recombinase onto DNA. Mutations in the BRCA2 gene increase the likelihood of developing breast and ovarian cancers because of a reduced capacity to repair DNA damage. The research proposed in this study aims to identify the critical parts of Orf responsible for helping RecA overcome the obstruction posed by single-stranded DNA binding proteins. We will employ biochemical assays to investigate how RecA polymer formation is influenced by the presence of Orf. In addition, we will study how RecA filament assembly is enhanced or disrupted in different contexts by visualising the proteins by electron microscopy. Finally, we will investigate how the Orf protein, which exists as a dual subunit ring, can open up like a clamp to bind single-stranded DNA. The results will give fresh insight into the molecular mechanisms of genetic recombination and the contribution this repair process makes in evading the onset of cancer.