UC Santa Barbara

The human genome endures insults from a variety of biological, chemical, and physical sources. Some of these insults result in severe lesions. One such lesion is the DNA double-strand break (DSB), in which the two strands of the DNA double helix are broken apart. This lesion can result from endogenous DNA damaging agents but can also be created during precision genome editing by targeted nucleases like CRISPR-Cas9. Regardless of the cause, human cells have multiple, competing DNA repair pathways by which to resolve these lesions; however, the cellular inputs that cells use to decide between these competing repair pathways remains elusive. My research objective was to monitor DNA repair factor recruitment to lesions in order to understand how the localization of DNA repair factors influences repair outcomes. In this work, I measure recruitment of both targeted nucleases and downstream repair factors by ChIP-seq, and I monitor changes to chromatin status and gene expression resulting from these activities using ATAC-seq and RNA-seq, respectively. In Chapter I, I describe the motivation behind the research projects and the development of a strand-specific ChIP-seq protocol to facilitate the measurement of the recruitment, localization, and polymerization of DNA repair proteins at DSBs. In Chapter II, I use this method to investigate the role of the FA-BRCA pathway during nuclease-induced DSB repair. I identify that the central player of the pathway, the FANCD2-FANCI heterodimer, localizes to DSBs and other open chromatin substates in a manner dependent on the activity of the FA Core Complex and ATM Kinase. I also find that FANCD2 coordinates chromatin remodeling, alters the spatial organization of other DNA repair factors such as BRCA1, and influences overall cell cycle progression. In Chapter III, I investigate the role of SFPQ during nuclease-induced DSB repair, and together with my co-authors, I identify that SFPQ does not localize to DSBs, but instead binds to the 5’ UTR of mRNA transcripts of RAD51 and its paralogs. In Chapter IV, I analyzed off-target transcriptional regulation events during CRISPRi screens, and I developed a methodology I termed POCKET-seq to characterize these binding events using gene ontology. In Chapter V, I discuss the implications of the work. Collectively, this work begins to untangle the molecular mechanisms underscoring the genetic, topological, and transcriptomic changes that occur during DNA repair and precision genome editing, which both rely on host DNA repair factors, chromatin remodelers, transcription factors, RNA-binding proteins, and splicing factors. Ultimately, this work has important implications for the development of cancer therapeutics and gene therapies for genetic disorders.