Structural Visualization of State Transition during Non-Homologous End-Joining (NHEJ)

Chen, Siyu

doi:https://doi.org/10.21985/n2-f308-hk59

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Structural Visualization of State Transition during Non-Homologous End-Joining (NHEJ)

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DNA double-strand breaks (DSBs) are caused by either endogenous agents or exogenous ionizing radiation and chemicals. Incorrect DSB repair can lead to undesired genome rearrangements. Homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways are two major DNA repair pathways that repair DSBs and maintain genome integrity. When homologous DNA is available as a template, HR uses it to direct error-free repairs, while NHEJ directly ligates the two broken DNA ends without any template. In immune cells, the V(D)J recombination pathway also uses NHEJ to assemble various immunoglobulin gene segments. While playing an important role in maintaining the stability and integrity of genomes, NHEJ is thought to be a ‘dirty’ DNA break repair pathway that is more error-prone compared to HR. It contributes to a large portion of chromosomal translocations in cells, making them particularly vulnerable to cancer-related mutations. It has been shown that ablation of NHEJ factors in DNA repair-deficient cell lines reduces genomic instability, and suppression of the NHEJ pathway kills cancer cells.NHEJ is initialized by a DSB recognition step, where Ku heterodimer (Ku70/80) recognizes and binds to the broken DNA ends. Upon binding to DNA, Ku recruits the DNA-dependent protein kinase (DNA-PKcs) to the repair loci and forms the DNA-PK holoenzyme. This holoenzyme then serves as the scaffold to recruit other core NHEJ factors to the repair site, including the core NHEJ factors (DNA ligase IV, XRCC4, XLF), appendix scaffolding factors (PAXX, APLF), nucleases (Artemis), polymerases (Pol μ and Pol λ) and other processing enzymes (PNKP, Tdp1/2). While the nucleases and polymerases are only recruited when end processing is needed prior to the end ligation, the core NHEJ factors are responsible for the final ligation step of the broken DNA ends and are therefore absolutely required in NHEJ. Successful DSB repair in NHEJ relies on the efficient bridging of two DNA ends, yet the detailed molecular mechanism that lies behind this pathway remains largely unclear. This project focuses on the key intermediate complexes in the NHEJ pathway and the main goal is to structurally visualize the mechanism of how NHEJ factors orchestrate in these key complexes as molecular machinery to repair DSB DNA. To achieve this goal, in vitro reconstitution in conjunction with single-particle cryo-electron microscopy (cryo-EM) were utilized to obtain the structure of five different states in NHEJ, including the Long-range (LR) complex, PAXX bound LR complex (PAXX-LR), ATP activated PAXX-LR complex (PAXX-ATP- LR), the Short-range complex (SR) and an alternative DNA-PKcs dimeric complex. Together, the key intermediates observed with Cryo-EM provide a comprehensive understanding of how DNA- PK holoenzyme bridge the two broken DNA ends in close proximity, recruit other core NHEJ factors, transit into the ligation state upon ATP activation and dissociation of DNA-PKcs. In addition, the results shed light on how different appendix NHEJ factors can be recruited by different mechanisms at different stages. In summary, direct visualization toward the overall architecture of key nucleoprotein assemblies during NHEJ can lead to the clarification of how these essential NHEJ factors orchestrate in such a complicated, multi-step DNA repair pathway. Understanding the molecular mechanism of this dominant DSB repair pathway in eukaryotic cells can also open up new directions toward cancer drug development and rational design of optimized gene-editing tools.

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