The role of ATM in the cell cycle control of V(D)J recombination

Lymphocyte development requires the assembly of diversified antigen receptor complexes generated by the genetically programmed V(D)J recombination event. Because germline DNA is cut, introducing potentially dangerous double-stranded breaks (DSBs) and rearranged prior to repair, its activity is limited to the non-cycling stages of the cell cycle, G0/G1. The potential involvement of a key mediator, Ataxia Telangiectasia Mutated or ATM, in the DNA damage response (DDR) and cell cycle checkpoints has been implicated in recombination, but its role is not fully understood. Thymic lymphomas from ATM deficient mice contain clonal chromosomal translocations involving the T-cell antigen receptor (TCR). A previous report found ATM and its downstream target p53 associated with V(D)J intermediates, suggesting the DDR senses recombination. In this study, we sought to understand the role of ATM in V(D)J recombination. Developing thymocytes from ATM deficient mice were analyzed according to the cell cycle to detect V(D)J intermediates. Examination of all TCR loci in the non-cycling (G0/G1) and cycling (S/G2/M) fractions revealed the persistence of intermediates in ATM deficient thymocytes, contrary to the wild-type in which intermediates are found only during G0/G1. Further analysis found no defect in end-joining of intermediates, nor were they detected in developed T-cells. Based upon the presence of persisting intermediates, the recombination initiating nuclease Rag-2 was examined; strict regulation limits it to G 0/G1. Rag-2 regulation was not affected by an ATM deficiency as Rag-2 expression remained contained within G0/G 1, indicating recombination is not continuous. To determine if an ATM deficiency affects recognition of V(D)J breaks, sites of recombination identified by a TCR locus or Rag expression were analyzed according to co-localization with a DDR factor phosphorylated immediately after DNA damage, phosphorylated H2AX (γH2AX). No differences in co-localization were found between the wild-type and ATM deficiency, demonstrating ATM deficient lymphocytes retain the ability to recognize DSBs. Together, these results suggest ATM is necessary in the cell cycle regulation of recombination but not essential for the identification of V(D)J breaks. ATM ensures the containment of intermediates within G0/G1 and maintains genomic stability of developing lymphocytes, emphasizing its fundamental role in preventing tumorigenesis.^