Low CDK Activity and Enhanced Degradation by APC/CCDH1 Abolishes CtIP Activity and Alt-EJ in Quiescent Cells

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 benefit from 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 the cell cycle phase, is at maximum in G(2)-phase, substantially reduced in G(1)-phase and almost undetectable in quiescent, G(0)-phase cells. The mechanism underpinning this regulation remains uncharacterized. Here, we compare alt-EJ in G(1)- and G(0)-phase cells exposed to ionizing radiation (IR) and identify CtIP-dependent resection as the key regulator. Low levels of CtIP in G(1)-phase cells allow modest resection and alt-EJ, as compared to G(2)-phase cells. Strikingly, CtIP is undetectable in G(0)-phase cells owing to APC/C-mediated degradation. The suppression of CtIP degradation with bortezomib or CDH1-depletion rescues CtIP and alt-EJ in G(0)-phase cells. CtIP activation in G(0)-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 suggest that suppression of mutagenic alt-EJ in G(0)-phase is a mechanism by which cells of higher eukaryotes maintain genomic stability in a large fraction of non-cycling cells in their organisms.

Automated summary

Alt-EJ is an error-prone DNA double-strand break repair pathway that is active when primary repair pathways, c-NHEJ and HR, are defective or fail. It depends on DNA end-resection, initiated by the CtIP/MRE11-RAD50-NBS1 complex and extended by EXO1 or BLM/DNA2 complex. Alt-EJ is regulated by the cell cycle phase, with maximum activity in G(2)-phase and reduced activity in G(1)-phase and quiescent G(0)-phase cells. CtIP-dependent resection is identified as the key regulator of Alt-EJ in G(1)- and G(0)-phase cells, with low levels of CtIP allowing modest resection and Alt-EJ in G(1)-phase cells and undetectable CtIP and Alt-EJ in G(0)-phase cells.