Abstract
DNA double-strand breaks (DSBs) are among the most toxic forms of DNA damage and pose a severe threat to genomic integrity. As such, the cell has evolved highly coordinated and complex mechanisms to repair such lesions. Improper repair of DSBs can lead to chromosomal duplication, chromosomal deletions, and chromosomal translocations, all of which are hallmarks of human cancers. Previous work in DNA damage has focused on the mechanistic characterization of how cells repair DNA DSBs, and my research builds on this foundation. In this work, I study repair of DNA DSBs made with the site-specific nucleases HO and CRISPR/Cas9 to show repair via single-stranded DNA, a common method of gene editing, utilizes an uncharacterized Rad51-independent DNA repair pathway that is dependent on Rad52, Srs2, and the MRX complex, but independent of other canonical repair proteins. We also find that Rad59 plays a significant role in the process by alleviating Rad51’s inhibition of Rad52 via the Rad51 homologs. Furthermore, genome-wide genetic screening suggests that this pathway also utilizes proteins that are traditionally components of nucleotide excision repair and telomere recombination. Additionally, I investigate the effect of heterologous templates during the more conventional DSB repair pathway, gene conversion. Here, we show that there is an inherent asymmetry in DSB repair, as correction of mismatches templated upstream of the DSB are mechanistically different than those templated downstream of the DSB. While the activity of polymerase ? is primarily responsible for incorporating mismatches into the recipient locus on the left side of the DSB, mismatches templated on the right side of the DSB are primarily corrected via components of the mismatch repair pathway (MMR). These corrections patterns, however, are heavily influenced by the nature of the DSB itself. Collectively, these results highlight the immense complexity of DNA DSB repair and offer insights into the mechanisms of DNA repair, as well as to the field of genome engineering.