Product of the surrounding : Location dependent effects on DNA double strand break responses

Abstract

Repair of DNA double strand breaks is required to sustain cell viability. In the last few decades, many factors have been identified that are critical for proper regulation of the DNA damage responses. Intriguingly, many of these factors are regulators of chromatin and nuclear architecture. However, we currently have a rather rudimentary understanding of the interplay between chromatin and DNA repair. For example, we do not fully understand how histone modifications and chromatin composition affect repair pathway preference. Also, we have little understanding of the mechanisms that ensure that the epigenetic landscape surrounding a break is properly restored after DNA repair. For this thesis, I investigated this interplay in more detail. Chapter 2, describes a role for the kinase Tlk2 in checkpoint recovery from a G2 DNA damage induced arrest. Following DNA repair, Tlk2 phosphorylates ASf1a to ensure proper deposition of histones. In the absence of histone deposition, G2 checkpoint recovery was defective due to decreased of expression of pro-mitotic genes. We found that downregulation of the p53 tumor suppressor rescues these defects. These results indicate that proper restoration of the chromatin is essential for G2 checkpoint recovery. In Chapter 3, we report on location-specific effects of damage in the rDNA and found that DNA breaks in the 45S rDNA repeat are liable to homologous recombination (HR). Previously, HR was considered to be error-free and therefore not detrimental to cells. However, we show that breaks in the 45S rDNA are toxic when repaired by HR, due to recombination between repeats. In addition, we identified SMC5 as a regulator of HR in rDNA repeats. In Chapter 4, we describe a system to generate location specific DNA breaks by Cas9. Using this system, we studied the cellular response to very low number of DNA breaks. We found that single breaks can signal through ATM and ATR to delay cell cycle progression. Perturbation of these delays led to excess DNA breaks in mitosis which resulted in genomic instability. In Chapter 5, we used the system described in Chapter 4 to study differences in cell fate in response to breaks in 18 different locations. We found that DNA break location dictates repair pathway usage. In addition, we demonstrated that these breaks display varying rates of proliferation, all of which is dependent on p53. Furthermore, we identified a subset of breaks on which DNA end resection activity is detrimental to cells. Taken together, these findings indicate that not all breaks display similar behavior in the context of cell fate and DNA repair.