Abstract
Chromosomal fragile sites are a cytogenetic phenomenon of genome instability that manifests in gaps or breaks on metaphase chromosomes. Diverse mechanisms are involved in their expression but all of them originate from the idea of replication impairment as the main driver for fragility. Cellular sensing of replication difficulties is crucial for correct duplication of the DNA to maintain genome integrity over generations. Untimely or defective sensing and repair at common fragile sites (CFSs) can result in major rearrangements, potentially leading to the development of cancer or other diseases. It has been proposed that cell-‐type specific CFSs give rise to cell-‐type specific diseases. Therefore, it is of special interest to better define and understand genomic regions that are prone to breakage.
In this PhD thesis a new approach to map CFSs genome-‐wide was established. This mapping method is not relying on cytogenetic analysis, instead using a FANCD2 ChIP-‐seq high-‐throughput approach. It enables the application of the method irrespective of the original tissue. FANCD2 enrichment sites were validated as bona fide CFSs in avian DT40 cells. To additionally elucidate genomic consequences of CFS expression, a HyTK mutation assay was established in this thesis. The fluctuation analysis-‐based HyTK counterselection assay demonstrated a correlation between mutational events and FANCD2 enrichment sites. The HyTK mutation assay can be a universal tool for the measurement of mutation rates in various cell systems.
With the identification and investigation of CFSs in avian DT40 cells, this study reveals the genome-‐wide evolutionary conservation of CFSs beyond the mammalian lineage for the first time. It opens the way for speculations on the beneficial existence of CFSs throughout the animal kingdom.
In this PhD thesis a new approach to map CFSs genome-‐wide was established. This mapping method is not relying on cytogenetic analysis, instead using a FANCD2 ChIP-‐seq high-‐throughput approach. It enables the application of the method irrespective of the original tissue. FANCD2 enrichment sites were validated as bona fide CFSs in avian DT40 cells. To additionally elucidate genomic consequences of CFS expression, a HyTK mutation assay was established in this thesis. The fluctuation analysis-‐based HyTK counterselection assay demonstrated a correlation between mutational events and FANCD2 enrichment sites. The HyTK mutation assay can be a universal tool for the measurement of mutation rates in various cell systems.
With the identification and investigation of CFSs in avian DT40 cells, this study reveals the genome-‐wide evolutionary conservation of CFSs beyond the mammalian lineage for the first time. It opens the way for speculations on the beneficial existence of CFSs throughout the animal kingdom.
| Originalsprog | Engelsk |
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| Forlag | Department of Biology, Faculty of Science, University of Copenhagen |
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| Antal sider | 150 |
| Status | Udgivet - 2015 |