H2AX prevents CtIP-mediated DNA end resection and aberrant repair in G1-phase lymphocytes

Abstract

DNA double stranded breaks (DSBs) are generated by the RAG endonuclease in all developing lymphocytes as they assemble antigen receptor genes1. DNA cleavage by RAG occurs only at the G1-phase of the cell cycle and generates two hairpin-sealed DNA (coding) ends that require nucleolytic opening prior to their repair by classical non-homologous end-joining (NHEJ)1–3. Although there are several cellular nucleases that could perform this function, only the Artemis nuclease is able to do so efficiently2, 3. Here we show, in vivo, that the histone protein H2AX prevents nucleases other than Artemis from processing hairpin-sealed coding ends; in the absence of H2AX, CtIP can efficiently promote the hairpin opening and resection of DNA ends generated by RAG cleavage. This CtIP-mediated resection is inhibited by γ-H2AX and by MDC-1, which binds to γ-H2AX in chromatin flanking DNA DSBs. Moreover, the ATM kinase activates antagonistic pathways that modulate this resection. CtIP DNA end resection activity is normally limited to cells at post-replicative stages of the cell cycle where it is essential for homology-mediated repair4, 5. In G1-phase lymphocytes, DNA ends that are processed by CtIP are not efficiently joined by classical NHEJ and the joints that do form frequently use micro-homologies and exhibit significant chromosomal deletions. Thus, H2AX preserves the structural integrity of broken DNA ends in G1-phase lymphocytes thereby preventing these DNA ends from accessing repair pathways that promote genomic instability.

Ataxia telangiectasia-mutated protein and DNA-dependent protein kinase have complementary V(D)J recombination functions

“…Conceivably, ATM and DNA-PKs phosphorylation might regulate RAG and thereby, affect SE ligation. Finally, it still is notable the SJs that do form in the absence of DNA-PKcs and ATM have increased junctional base pair loss, suggesting a potential overlapping function in end protection, reminiscent of that recently described for H2AX in the context of unjoined CEs and SEs (9,39). Indeed, it is quite possible that the overlapping functions of DNA-PKcs and ATM kinase activities might promote SJ formation through phosphorylation of several different substrates.…”

Functional redundancy between the XLF and DNA-PKcs DNA repair factors in V(D)J recombination and nonhomologous DNA end joining

“…In addition, unjoined CEs and SEs generated during attempted V(D)J recombination in XLF/H2AX and XLF/53BP1 double-deficient pro-B cells are highly resected, consistent with a role for H2AX and 53BP1 in end protection (34)(35)(36). It is notable that ATM deficiency or treatment with ATM inhibitors rescues the end resection, but not the V(D)J joining phenotype of XLF/H2AX or XLF/53BP1 doubledeficient pro-B lines (31,32); this is consistent with ATM having both a role in end joining and in activating the CtIP nuclease for end resection (34). Potential roles for XLF in end protection have been speculated but not tested.…”

“…S1. double mutants might reflect some unknown role of DNA-PKcs, similar to that found for ATM (34), in activating resection. However, we also found no increased resection of unjoined CEs that accumulate in hairpin form in XLF Δ/Δ Artemis −/− and as opened ends in XLF Δ/Δ XRCC4 −/− v-Abl transformed pro-B lines (Fig.…”

CtIP-mediated resection is essential for viability and can operate independently of BRCA1

“…In contrast, extensive resection during class switching in 53BP1 2/2 cells is ATM-independent (Yamane et al, 2013), and is mediated by both CtIP and EXO1 . Similarly, unrepaired G1-phase breaks during V(D)J recombination are processed by CtIP (Helmink et al, 2011). These results suggest that, whereas CtIP promotes nucleolytic processing of DSBs in all cell cycle phases, other factors that stimulate or antagonize the extension of resected tracks may be distinct and regulated in a cell cycle-dependent manner.…”