Molecular Mechanisms of Flexibility in Nonhomologous End Joining Public Deposited

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Last Modified
  • March 21, 2019
Creator
  • Conlin, Michael
    • Affiliation: School of Medicine, Curriculum in Genetics and Molecular Biology
Abstract
  • DNA double strand breaks (DSBs) are highly toxic DNA lesions that play a critical role in human health and disease. The ability to repair these lesions is essential in all kingdoms of life, and in mammals is primarily attributed to the nonhomologous end joining (NHEJ) pathway. NHEJ faces a unique challenge: unlike other forms of DNA damage, DSBs are structurally heterogeneous, varying wildly in end chemistry. To address this problem, NHEJ has evolved uniquely flexible enzymes: DNA polymerases and a DNA ligase that can act on a remarkable variety of substrates, much more so than their counterparts in other pathways. The mechanistic basis of this flexibility, and its significance to biological repair, are unknown. DNA Ligase IV (LIG4) is the only human DNA ligase that participates in NHEJ, and the only one that can efficiently ligate ends across gaps, or with terminal mispairs. We show by single-molecule analysis that terminal mispairs lead NHEJ complexes to mobilize DNA ends and thereby sample more end alignments. This flexibility is what allows LIG4 to join such ends, since pairing flexibility and ligation both require a LIG4-specific structural motif, insert1. Our work showed that pairing flexibility is what enables LIG4 to tolerate a chemically diverse array of substrates, and that this tolerance is essential for cells to survive exogenous DNA damage such as ionizing radiation. NHEJ employs two uniquely flexible polymerases to prepare ends for ligation: DNA polymerase μ (pol µ) and terminal deoxynucleotidyl transferase (TdT). These enzymes act on noncanonical substrates that other polymerases cannot engage. We show these polymerases primarily incorporate ribonucleotides (RNA), not deoxynucleotides (DNA), during NHEJ, both during repair of chromosome breaks made by Cas9 and during V(D)J recombination. These ribonucleotides facilitate NHEJ by enabling ligation of ends with adjacent mispairs, and even single strand ligation. Supplementing cells expressing TdT with deoxynucleotides thus blocks repair of Cas9-induced breaks, while ribonucleotide supplementation can improve Cas9-directed mutagenesis. Our results indicate cellular NHEJ often involves transiently embedded ribonucleotides, which promote flexibility in repair at the cost of more fragile intermediates.
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  • In Copyright
Advisor
  • Ahmed, Shawn
  • Sekelsky, Jeff
  • Ramsden, Dale
  • Williams, Scott
  • Erie, Dorothy
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill Graduate School
Graduation year
  • 2018
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