Student Theses and Dissertations

Date of Award

2009

Document Type

Thesis

RU Laboratory

de Lange Laboratory

Keywords

DNA repair, DNA lesions, DNA ends, non-homologous end-joining, microtubules, 53BP1

Abstract

When a double-stranded break (DSB) occurs in mammalian genomes, the local chromatin is altered through the modification of histones (notably the phosphorylation of H2AX) and the binding of DNA damage response factors (e.g. MDC1, 53BP1). Although several lines of evidence have pointed to a role for some of these factors in DSB repair through non-homologous end-joining (NHEJ), the mechanism of their contribution has not been established. To study the regulation of NHEJ, we have used as a model system dysfunctional telomeres, which are uncapped by the removal of the shelterin component, TRF2. As a consequence of TRF2 loss, deprotected chromosome ends trigger a sequence of events normally activated by the presence of DSBs. These include the instigation of ATMmediated activation of cell cycle checkpoints and the accumulation of DNA damage response factors at the telomeric chromatin. In addition, the NHEJ pathway repairs deprotected telomeres to generate chromosome end-to-end fusions. We have examined the roles of the Mre11/Rad50/NBS1 (MRN) complex, H2AX, MDC1, and 53BP1 in the NHEJ of dysfunctional telomeres. We have demonstrated that among these factors, 53BP1 is required for the fusion of telomeres, whereas the MRN complex, H2AX, and MDC1 only stimulate the efficiency of the repair process, most likely by mediating the recruitment of 53BP1 to uncapped chromosome ends. Furthermore, we have revealed the mechanism by which 53BP1 acts. We have shown that upon deprotection, telomeres become more dynamic and explore larger territories in a 53BP1-dependent manner. Faster mobility of DNA ends increases the chance that dysfunctional telomeres, which are uniformly scattered throughout the nucleus, will find one another and fuse. We have proposed that the dynamic behavior of DNA ends may be required to promote long-distance repair in general, and that it may play a role in other instances of NHEJ, such as during recombination in the immunoglobulin genes, where the DNA ends are initially at a distance. Furthermore, we have shown that the mechanism that promotes the mobility of uncapped chromosome ends requires microtubules. This finding suggests an unprecedented role for microtubules in the process of DNA repair in mammalian interphase cells. Moreover, it points to the existence of a trans-nuclear envelope bridge between damaged chromatin and cytoplasmic microtubules. Accordingly, our data indicate that mobility depends on the acetylation status of chromatin, signifying that specific chromatin modifications are involved in establishing that connection. Finally, we have preliminary evidence that the dynamic process that we have uncovered might play a role in the repair of all DNA lesions. We speculate that a microtubule-dependent chromatin mobility provides a proofreading mechanism preventing HDR between non-sister chromatids, possibly by physically pulling apart inappropriate connections. Overall, this thesis presents a novel view on how the dynamic behavior of DNA ends might be required for efficient and accurate repair of DNA lesions.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/233

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