Mapping DNA repair through a rugged landscape: A DNA sequencing approach to study the effect of chromatin context on double-strand break repair

The human genetic information is coded in DNA molecules that if stretched would be more than two meters long. However, the DNA fits in the nucleus, a sphere of 10 micrometers. To accommodate two meters of DNA in the nucleus, DNA in human cells interacts with proteins in a structure called chromatin that adopts different conformations in human cells. A key feature of chromatin is that it constantly interplays with other processes occurring in the cell nucleus. DNA repair is one of these processes and is essential for cell survival. This is because the DNA in human cells often breaks due to physical or chemical stresses, and failure to repair breaks predispose humans to develop diseases like cancer. Even though the interplay between DNA repair and chromatin is well described, the molecular mechanism behind this interplay is poorly understood.

In this thesis, we describe the development and use of novel experimental and computational tools to systematically study the interplay between DNA repair and chromatin proteins mainly using high-throughput DNA sequencing technologies. In Chapter 2, we review the latest high-throughput sequencing technologies that allow for the study of DNA repair across the entire genome. Using fluorescence-based technologies as counterweights, we highlight new opportunities and limitations of highthroughput DNA sequencing. Because we extensively use the reviewed high throughput sequencing technologies throughout the thesis, this chapter aims to serve as a second introductory chapter of this thesis. In Chapter 3, we systematically knock out DNA repair proteins and measure pathway balance in multiple chromatin contexts in parallel. By doing so, we identify dozens of DNA repair proteins that regulate MMEJ:NHEJ balance in a chromatin contextdependent manner. Furthermore, we show that chromatin context dependencies of DNA repair proteins influence how human tumors accumulate mutations throughout the genome. In Chapter 4, we apply the same experimental framework to identify epigenetic drugs that affect Cas9 editing in a chromatin context dependent manner, and we find that HDAC inhibitors increase CRISPR/Cas9 editing frequency in heterochromatin. Because many DNA repair and chromatin proteins favor MMEJ preferentially in LADs, we also study how genome-nuclear lamina interactions change during DSB repair. In Chapter 5, we show that DSBs induce detachment of megabase-sized chromatin regions from the nuclear lamina in a γH2AX-dependent manner. Finally, in Chapter 6 we discuss the main findings of this thesis with a focus on the implications of chromatin-context dependencies of DNA repair proteins and their applicability in DNA repair.