Theses Doctoral

Homology-Directed Repair of One- and Two-Ended DNA Double-Strand Breaks

Kimble, Michael Taylor

DNA double-strand breaks (DSBs) are one of the most dangerous lesions cells encounter, given that DSBs can lead to genomic instability and cell death if not repaired properly. Cells have two primary pathways to repair DSBs: Homologous recombination (HR) and nonhomologous end-joining (NHEJ). HR is the high-fidelity branch of the DSB repair pathway since it employs a process of homology search and synthesis from a homologous template. The homology search is carried out by ssDNA that is generated on either side of the DSB by end resection. End resection occurs via a two-step mechanism involving resection initiation, followed by long-range resection. Previous work has revealed that long-range resection is dispensable for some cases of HR; however, it is currently unclear why the requirement for long-range resection is context- dependent. Furthermore, it is not completely clear how the mechanisms of HR, including requirements for long-range resection, apply to single-ended DSBs (seDSBs) arising during replication. Therefore, we defined the role of long-range resection in two-ended DSB repair in different chromosomal contexts. We also established a Cas9 nickase (Cas9n) system to study seDSB repair and defined genetic requirements for repair.

To study the requirement for long-range resection in HR, we employed inter- and intrachromosomal genetic recombination assays in haploid yeast. We found that long-range resection is required for interchromosomal HR, but not for intrachromosomal HR. This difference is linked to the observation that the DNA damage checkpoint, which is deficient in the absence of long-range resection, is activated in interchromosomal HR, but not intrachromosomal HR. The DNA damage checkpoint has also previously been implicated in promoting chromosome mobility. Therefore, we reason that the requirement for long-range resection in interchromosomal repair is due to a need to activate the DNA damage checkpoint and chromosome mobility, specifically during slower repair events.

To study seDSB repair, we implemented Cas9n, which creates nicks that can cause replication fork collapse. We demonstrated that expression of Cas9n with an efficient gRNA can induce replication fork collapse and that repair of these seDSBs breaks is dependent on the HR machinery. A genome-wide screen using Cas9n revealed a requirement for replication-coupled nucleosome assembly (RCNA) in repair of seDSBs, specifically in replication origin-deplete regions of the genome. Consistent with the model of seDSB repair, we found that Cas9n-induced seDSBs preferentially undergo sister chromatid recombination. This preference was altered in the absence of Mre11, which we hypothesize is due to a role of MRX in sister chromatid tethering. Altogether, the results presented in this thesis offer a different perspective on the role of long-range resection in two-ended DSB repair and establish a Cas9n-based system to better study single-ended DSB repair.


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More About This Work

Academic Units
Biological Sciences
Thesis Advisors
Symington, Lorraine S.
Ph.D., Columbia University
Published Here
July 19, 2023