ATM antagonizes NHEJ proteins assembly and DNA-ends synapsis at single-ended DNA double strand breaks

Abstract

Two DNA repair pathways operate at DNA double strand breaks (DSBs): non-homologous end-joining (NHEJ), that requires two adjacent DNA ends for ligation, and homologous recombination (HR), that resects one DNA strand for invasion of a homologous duplex. Faithful repair of replicative single-ended DSBs (seDSBs) is mediated by HR, due to the lack of a second DNA end for end-joining. ATM stimulates resection at such breaks through multiple mechanisms including CtIP phosphorylation, which also promotes removal of the DNA-ends sensor and NHEJ protein Ku. Here, using a new method for imaging the recruitment of the Ku partner DNA-PKcs at DSBs, we uncover an unanticipated role of ATM in removing DNA-PKcs from seDSBs in human cells. Phosphorylation of DNA-PKcs on the ABCDE cluster is necessary not only for DNA-PKcs clearance but also for the subsequent MRE11/CtIP-dependent release of Ku from these breaks. We propose that at seDSBs, ATM activity is necessary for the release of both Ku and DNA-PKcs components of the NHEJ apparatus, and thereby prevents subsequent aberrant interactions between seDSBs accompanied by DNA-PKcs autophosphorylation and detrimental commitment to Lig4-dependent end-joining.

DNA folds threaten genetic stability and can be leveraged for chemotherapy

“…133 In addition, ATM stimulates HR by phosphorylating several HR factors, such as BRCA2, EXO1 and BLM, and NHEJ factors such as XRCC4, XRCC4-like factor (XLF) and DNA-PKcs. [134][135][136][137] ATM also plays a crucial role in enforcing the cell cycle checkpoints through activation by phosphorylation of CHK2, which in turn activates p53, a key apoptosis regulator. 68 53BP1 is recruited to nucleosomes with specific histone modifications: Ring Finger Protein 168 (RNF168)-ubiquitylated H2AK15 and dimethylated H4K20; 138 where it functions as a central determinant in the repair pathway choice made at DSB by promoting the recruitment of the shieldin complex, 139 thereby limiting DNA end resection by BRCA1/BARD1, which exposes ssDNA to be repaired by HR.…”

The Safe Path at the Fork: Ensuring Replication-Associated DNA Double-Strand Breaks are Repaired by Homologous Recombination

“…Thus, the overhanging ends in early fork regression intermediates are intrinsically protected from cNHEJ, and end-protection by RAD51 and other factors appears to reinforce cNHEJ suppression. In addition, ATM promotes dissociation of DNA-PK from seDSBs, suppressing cNHEJ ( Britton et al, 2020 ). In the case of fork cleavage by either MUS81-EME2 or EEPD1 (or fork collapse at nicks), the initial state is a blunt, or nearly blunt seDSB, i.e., an excellent cNHEJ substate.…”

XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase

“…In addition, loss of XAB2 led to a significant increase in Ku80 foci irrespective of the increase in γH2AX foci, as showed by the difference between the intercepts of the shXAB2 and shCTRL regression lines (** P -value = 0.00146, Figure 3D ), consistent with the notion that XAB2 prevents Ku accumulation at seDSB termini. ATM defines one pathway for Ku release at seDSBs induced by CPT ( 11 , 71 ). In agreement with these studies, inhibition of ATM kinase activity using the ATM inhibitor (ATMi) KU-55933 ( 72 ) also resulted in increased Ku80 foci formation in TMZ-treated cells (Figure 3A ).…”