Overcoming obstacles to nonhomologous end joining repair of chromosome double strand breaks
Year: 2019
Type: dissertation
Abstract: Protein occlusions and oxidative nucleotide damages that flank chromosome double strand breaks are serious obstacles to nonhomologous end joining (NHEJ) since they each directly impact a specific step of this repair pathway. Because NHEJ requires the Ku heterodimer (a ring protein requiring 360o of access for binding) to recognize DNA ends, this pathway would seem particularly sensitive to DNA end-occlusions. End-associated nucleotide damages would also seemly present a significant obstacle to NHEJ as they specifically inhibit XRCC4-LigaseIV, and their proximity to DNA ends precludes their removal by traditional base excision repair enzymes. Surprisingly, NHEJ is able to overcome these obstacles. I have shown that despite the potential steric conflicts, Ku readily binds protein-occluded DNA ends. Moreover, this very protein, predicted to be inhibited by end-associated protein obstructions, may specifically limit NHEJ's need for ATP-dependent chromatin remodeling as Ku readily evicts weakly bound DNA-associated proteins and peels DNA from strongly positioned nucleosomes. In the case of end-associated nucleotide damages, NHEJ employs a large repertoire of processing factors, some displaying unique activity, to specifically remove, repair, or bypass these complex lesions. My work identifies a new part of this repertoire: Ku is novel AP lyase tailored to remove end-associated abasic sites and thereby facilitate joining of these complex breaks. Ku efficiently cleaves the phosphodiester backbone 3' of end-associated abasic sites, precisely removing damage in a context unfavorable to other known lyases. Finally, my work suggests NHEJ ensures difficult breaks containing incompatible ends are repaired. This involves complementing the above processing based ligation with an additional, low fidelity ligation mechanism. XLF/Cernunnos, the key factor establishing this subpathway, functions to clamp XRCC4-LigaseIV to DNA ends and thereby stimulates LigaseIV's ability to ligate mismatched overhangs. Consequently, this activity allows NHEJ to join difficult DNA ends, potentially incorporating damaged nucleotides or mismatches into an intact chromosome, where they can be repaired without further risk of DNA translocation. Taken together, these unique activities highlight NHEJ's flexibility and sophistication in overcoming obstacles to preserve genome integrity.
DOI: 10.17615/q729-rm82
Author: Roberts, Steven Andrew
Institution: University of North Carolina at Chapel Hill
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2246967790