The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies

Abstract

Non-homologous end-joining (NHEJ), the major repair pathway for DNA double-strand breaks (DSB) in mammalian cells, employs a repertoire of core proteins, the recruitment of which to DSB-ends is Ku-dependent. Lack of either of the core components invariably leads to a repair deficiency. There has been evidence that an alternative end-joining operates in the absence of the core components. We used chromosomal reporter substrates to specifically monitor NHEJ of single I-SceI-induced-DSB for detailed comparison of classical and alternative end-joining. We show that rapid repair of both compatible and non-compatible ends require Ku-protein. In the absence of Ku, cells use a slow but efficient repair mode which experiences increasing sequence-loss with time after DSB induction. Chemical inhibition and PARP1-depletion demonstrated that the alternative end-joining in vivo is completely dependent upon functional PARP1. Furthermore, we show that the requirement for PARP1 depends on the absence of Ku but not on DNA-dependent protein kinase (DNA-PKcs). Extensive sequencing of repair junctions revealed that the alternative rejoining does not require long microhomologies. Together, we show that mammalian cells need Ku for rapid and conservative NHEJ. PARP1-dependent alternative route may partially rescue the deficient repair phenotype presumably at the expense of an enhanced mutation rate.

Repair of Double-Strand Breaks by End Joining

“…Like c-NHEJ, alt-NHEJ demands DSB synapsis. PARP-1 has been implicated in this function in alt-NHEJ, reminiscent of the role of DNA-PKcs in c-NHEJ (Audebert et al 2008;Robert et al 2009;Mansour et al 2010). Not only is alt-NHEJ independent of DNA-PK, but also it is suppressed by Ku, implying a competition of factors for DSB ends that is typically won by c-NHEJ (Audebert et al 2004;Wang et al 2006).…”

Poly-ADP-Ribose Polymerase as a Therapeutic Target in Pediatric Diffuse Intrinsic Pontine Glioma and Pediatric High-Grade Astrocytoma

“…PARP1 has been shown to be involved in delaying the replication fork progression in homologous recombination (HR)-proficient DNA damaged cells and in alternative pathways of nonhomologous end joining (NHEJ; refs. [21][22][23]. PARP inhibitors are synthetically lethal in BRCA1/2-mutated tumors with DNA repair deficiencies and have been shown to be effective when combined with DNAdamaging agents in isogenic BRCA1/2-deleted cell lines and BRCA1-deficient mouse models (24)(25)(26)(27).…”

Molecular Pathways: How Can BRCA-Mutated Tumors Become Resistant to PARP Inhibitors?

“…Despite its interaction with XRCC1, PARP1 is not required for base excision repair and it has in fact been shown that recruitment of XRCC1 and OGG1 is not blocked by PARP inhibition (13). The current view is that PARP1 plays an important role in multiple DNA repair pathways including the alternative non-homologous end-joining (NHEJ) pathway (12,(14)(15)(16) and prevents excessive homologous recombination. The increase in sister chromatid exchange in PARP1 knockout mice and the accumulation of RAD51 foci in PARP1-deficient or PARP inhibitortreated cells (17) are consistent with this model.…”