FIDELITY OF DNA DOUBLE-STRAND BREAK REPAIR BY NONHOMOLOGOUS END JOINING

DNA stores the genetic information essential for all life as we know it, but it is constantly assaulted with damage. To survive, cells must maintain myriad DNA repair pathways addressing this damage. Chromosomal double-strand breaks are the most toxic of all damage types and can lead to mutations, cancer, and cell death if left unrepaired. Cells rely on three pathways to combat double-strand breaks: nonhomologous end joining, homologous recombination, and polymerase theta-mediated end joining. This dissertation broadly studies double-strand break repair, observing all pathways in collaboration (or competition) when a cell encounters chromosome breaks. It further describes key mechanistic features of nonhomologous end joining that confer fidelity and adaptability during the processing of diverse double-strand break ends. First, I analyze the independent contributions of double-strand break repair pathways at in a time-resolved approach, including the kinetics of chromosome breakage, the capture of transient repair intermediates, and the mutation patterns arising from the individual pathways. I further analyze repair spectra in contexts of genetic perturbation and small molecule inhibition of key players in the end joining pathways. Next, I study ribonucleotide incorporation by nonhomologous end joining polymerases. Using extrachromosomal substrates and chromosome breaks through CRISPR-Cas9 or an inducible V(D)J recombination system, I dissect the specific contributions of ribonucleotide incorporation to the efficiency and accuracy of double-strand break repair and what factors regulate their use. Finally, I address the mechanisms by which nonhomologous end joining processes and repairs the individual strands of a double-strand break. Using novel assay designs, I show that the nonhomologous end joining machinery adapts to a variety of end structures and sequences, not just between distinct double-strand breaks, but also between the two strands of a single broken DNA molecule. Throughout this dissertation, I develop a comprehensive picture of double-strand break processing. I define mechanisms of nonhomologous end joining by which it addresses diverse double-strand break end structures to promote high fidelity repair. These findings help further define processes that promote the genome maintenance essential for cell survival.