Pathways of NHEJ at Dysfunctional Telomeres and Their Resolution

Shelterin is a multiprotein complex that prevents DNA damage signaling at chromosome ends. In its absence, DNA repair pathways are activated that can promote the fusion of dysfunctional telomeres resulting in chromosomal instability. The work presented here aims to understand how telomeres are protected from these pathogenic repair pathways. The first part of this thesis is focused on the DNA damage response factor 53BP1, a key regulator in double strand break (DSB) repair pathways in mammalian cells. By influencing key regulatory events at and near DNA ends, 53BP1 plays an important role in the decision between non-homologous endjoining (NHEJ) and homologous recombination (HR). Telomeres lacking the shelterin protein TRF2 have proven a versatile system for studying 53BP1 in DSB repair since 53BP1 protects dysfunctional telomeres from resection and promotes their mobility. Analysis of separation of function mutants of 53BP1 has identified the domain that is responsible for promoting the mobility of DSBs. Cells expressing 53BP1ΔMOB showed reduced levels of NHEJ at dysfunctional telomeres and damage foci roamed a smaller part of the nucleus. But which 53BP1 interacting factors are responsible for mediating the increased mobility remains unclear. The second part of this thesis aims to elucidate the DNA repair pathways that are activated when the shelterin component TRF1 is absent. The results provide tantalizing evidence that sister telomeres can fuse via the alternative- NHEJ (a-NHEJ) pathway when replication forks reach the end of the chromosome in cells that are deficient in TRF1. To prevent chromosome instability, mammalian cells appear to employ the Holliday Junction resolvase Gen1, which is capable of cleaving cruciform DNA structures that can be formed when telomeres fuse. The deletion of Gen1 from TRF1 null cells resulted in the accumulation of chromatin bridges and isochromosomes, indicative of genomic instability. The results presented here are of relevance to studies of human cancer cells since the presence of critically short telomeres induces sister telomere fusions via a-NHEJ. It is possible that replication stress underlies these fusion events and that Gen1 has evolved as a mechanism to counteract the consequences of chromosomal fusion events at telomeres

53BP1 and the LINC Complex Promote Microtubule-Dependent DSB Mobility and DNA Repair

SummaryIncreased mobility of chromatin surrounding double-strand breaks (DSBs) has been noted in yeast and mammalian cells but the underlying mechanism and its contribution to DSB repair remain unclear. Here, we use a telomere-based system to track DNA damage foci with high resolution in living cells. We find that the greater mobility of damaged chromatin requires 53BP1, SUN1/2 in the linker of the nucleoskeleton, and cytoskeleton (LINC) complex and dynamic microtubules. The data further demonstrate that the excursions promote non-homologous end joining of dysfunctional telomeres and implicated Nesprin-4 and kinesins in telomere fusion. 53BP1/LINC/microtubule-dependent mobility is also evident at irradiation-induced DSBs and contributes to the mis-rejoining of drug-induced DSBs in BRCA1-deficient cells showing that DSB mobility can be detrimental in cells with numerous DSBs. In contrast, under physiological conditions where cells have only one or a few lesions, DSB mobility is proposed to prevent errors in DNA repair.PaperCli