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.