The suppression of alt-EJ in quiescent G0-phase cells is caused by enhanced APC/CCDH1-mediated CtIP degradation and low CDK levels

Abstract

Abstract Alt-EJ is an error-prone DNA double-strand break (DSBs) repair pathway coming to the fore when first-line repair pathways, c-NHEJ and HR, are defective or fail. It is thought to depend on DNA end-resection: a process whereby 3’ single-stranded DNA-tails are generated, initiated by the CtIP/MRE11-RAD50-NBS1 (MRN) complex and extended by EXO1 or the BLM/DNA2 complex. The connection between alt-EJ and resection remains incompletely characterized. Alt-EJ depends on cell cycle phase, is at maximum in G2-phase, substantially reduced in G1-phase and almost undetectable in quiescent, G0-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G1- and G0-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G1-phase allow modest resection and alt-EJ as compared to G2-phase cells. Strikingly, CtIP is undetectable in G0-phase cells owing to APC/C mediated degradation. Suppression of CtIP degradation with bortezomib or CDH1 knockdown rescues CtIP and alt-EJ in G0-phase cells. CtIP activation in G0-phase cells also requires CDK-dependent phosphorylation by any available CDK, but is restricted to Cdk4/6 at the early stages of the normal cell cycle. We propose that suppression of alt-EJ in G0-phase enhances genomic stability in the large non-cycling cell fraction of higher eukaryotes.