| Summary: | <p>Centromeres are structurally essential regions of the eukaryotic genome, acting as platforms for kinetochore establishment and microtubule attachment during chromosome segregation. Despite their essential function, centromeres are implicated during genome instability, as DNA breaks and rearrangements originating at centromeres are frequently observed in human disease.</p>
<p>In this thesis, I first investigate the innate DNA fragility of centromeres through two independent approaches. I perform computational analyses of publicly available next-generation sequencing datasets, revealing a striking incidence of spontaneous DNA single-strand breaks at human centromeres. Through the development of a novel single-cell imaging assay to directly assess DNA breaks at repetitive regions of the genome including centromeres, I confirm this observation and further investigate potential sources of spontaneous centromere fragility. I also provide evidence that centromeric DNA breaks are surprisingly refractory to canonical DNA damage signalling mechanisms, raising important questions regarding the detection and repair of DNA breaks at centromeres.</p>
<p>Secondly, building on a growing body of evidence suggesting that centromeres are unusually prone to spontaneous recombination, I examined the role of the repair pathway homologous recombination (HR) at human centromeres. I demonstrate that the central mammalian recombinase RAD51 suppresses DNA break accumulation in a manner dependent on its recombination activity, providing molecular evidence for the recombination processes proposed to occur at human centromeres.</p>
<p>Finally, I show that RAD51 and its canonical regulator BRCA2 are intimately linked to the maintenance of centromere identity, defined by the centromere-specific epigenetic marker CENP-A, providing insight into a potential function of innate centromere fragility.</p>
<p>Based on the results presented in this thesis, I propose that human centromeres are innately susceptible to spontaneous DNA breaks, which are subsequently targeted for recombination-based repair. These findings shed light on several questions ranging from centromere-derived genome instability in human disease to our fundamental understanding of genome evolution.</p>
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