Multifunctional properties of Nej1XLF C-terminus promote end-joining and impact DNA double-strand break repair pathway choice

A DNA double strand break (DSB) is primarily repaired by one of two canonical pathways, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ requires no or minimal end processing for ligation, whereas HR requires 5 end resection followed by a search for homology. The main event that determines the mode of repair is the initiation of 5 resection because if resection starts, then NHEJ cannot occur. Nej1 is a canonical NHEJ factor that functions at the cross-roads of repair pathway choice and prior to its function in stimulating Dnl4 ligase. Nej1 competes with Dna2, inhibiting its recruitment to DSBs and thereby inhibiting resection. The highly conserved C-terminal region (CTR) of Nej1 (330- 338) is important for two events that drive NHEJ, stimulating ligation and inhibiting resection, but it is dispensable for end-bridging. By combining nej1 point mutants with nuclease-dead dna2-1, we find that Nej1-F335 is essential for end-joining whereas V338 promotes NHEJ indirectly through inhibiting Dna2-mediated resection.

The (Lack of) DNA Double-Strand Break Repair Pathway Choice During V(D)J Recombination

DNA double-strand breaks (DSBs) are highly toxic lesions that can be mended via several DNA repair pathways. Multiple factors can influence the choice and the restrictiveness of repair towards a given pathway in order to warrant the maintenance of genome integrity. During V(D)J recombination, RAG-induced DSBs are (almost) exclusively repaired by the non-homologous end-joining (NHEJ) pathway for the benefit of antigen receptor gene diversity. Here, we review the various parameters that constrain repair of RAG-generated DSBs to NHEJ, including the peculiarity of DNA DSB ends generated by the RAG nuclease, the establishment and maintenance of a post-cleavage synaptic complex, and the protection of DNA ends against resection and (micro)homology-directed repair. In this physiological context, we highlight that certain DSBs have limited DNA repair pathway choice options.

RB Regulates DNA Double Strand Break Repair Pathway Choice by Mediating CtIP Dependent End Resection

Inactivation of the retinoblastoma tumor suppressor gene (RB1) leads to genome instability, and can be detected in retinoblastoma and other cancers. One damaging effect is causing DNA double strand breaks (DSB), which, however, can be repaired by homologous recombination (HR), classical non-homologous end joining (C-NHEJ), and micro-homology mediated end joining (MMEJ). We aimed to study the mechanistic roles of RB in regulating multiple DSB repair pathways. Here we show that HR and C-NHEJ are decreased, but MMEJ is elevated in RB-depleted cells. After inducing DSB by camptothecin, RB co-localizes with CtIP, which regulates DSB end resection. RB depletion leads to less RPA and native BrdU foci, which implies less end resection. In RB-depleted cells, less CtIP foci, and a lack of phosphorylation on CtIP Thr847, are observed. According to the synthetic lethality principle, based on the altered DSB repair pathway choice, after inducing DSBs by camptothecin, RB depleted cells are more sensitive to co-treatment with camptothecin and MMEJ blocker poly-ADP ribose polymerase 1 (PARP1) inhibitor. We propose a model whereby RB can regulate DSB repair pathway choice by mediating the CtIP dependent DNA end resection. The use of PARP1 inhibitor could potentially improve treatment outcomes for RB-deficient cancers.