Ku Regulates the Non-Homologous End Joining Pathway Choice of DNA Double-Strand Break Repair in Human Somatic Cells

Abstract

The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer-dependent or “classic” NHEJ (C-NHEJ) pathway and an “alternative” NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.

Nonhomologous end joining drives poly(ADP-ribose) polymerase (PARP) inhibitor lethality in homologous recombination-deficient cells

“…The end-joining reporter plasmid pEGFP-Pem1-Ad2 (provided by E. Hendrickson, University of Minnesota, Minneapolis, MN) (Fig. 3A) was used as previously described (16,30,31). Further details are provided in SI Materials and Methods.…”

“…Thus, PARP inhibitors induce DNA-PK activation, as manifested by phosphorylation of DNA-PK substrates and formation of foci containing phosphorylated DNA-PKcs, only in BRCA2-deficient PEO1 cells and not BRCA2-positive PEO4 cells. To directly measure the effect of PARP inhibition on NHEJ activity in vivo, we used a validated reporter assay (16,30,31) (Fig. 3A).…”

“…Using an assay for MMEJ (31,33) (Fig. S5A) that readily detected MMEJ in DNA-PKcs-deficient M059J cells (Fig.…”

DNA double strand break repair pathway choice: a chromatin based decision?

“…Many recent studies have put effort into identifying the key factors that could regulate the switch toward one or the other pathway. For example, factors inhibiting resection such as the Ku heterodimer, 12 53BP1 and its effectors RIF1 (reviewed in 13 ) or PTIP 14 will favor NHEJ. On the other hand, CtIP 15 and the MRE11 nuclease 16 promote resection and thus HR.…”

Repair of Accidental DNA Double-Strand Breaks in the Human Genome and Its Relevance to Vector DNA Integration

“…Thus, how and which pathway is chosen for repair of a DSB(s) has been a critical issue in the DNA repair field, and there has been a debate [4]. Recent evidence suggests that Ku-bound DSBs, where end resection does not occur, are directed to NHEJ, while end-resected DSBs, to which Ku cannot bind, are channeled to HR (or alternative end-joining) [20][21][22][23][24]. Thus, in addition to the end binding protein Ku, various factors that regulate end resection are involved in DSB repair pathway choice [16,[25][26][27][28][29][30].…”