Mario Martinez

THE ROLE OF DOWNSTREAM ATM PROTEINS IN DOUBLE STRAND BREAK REPAIR DURING V(D)J RECOMBINATION. Mario Martinez¹, Andrea Bredemeyer², Barry Sleckman²; Department of Biomedical Engineering, Washington University, St. Louis, MO¹; Department of Pathology and Immunology, Washington University; St. Louis, MO.²

In order to create a repertoire of different antigen receptors, lymphocytes need to create DNA double strand breaks (DSBs) and repair them in a process known as V(D)J recombination. During the G1 Phase of the cell cycle, recombinase activating genes 1 and 2 (RAG-1 and RAG-2) create these DSBs in DNA, which are composed of two hairpin coding ends and two blunt signal ends. After DSBs are created, the non-homologous end joining (NHEJ) DSB repair proteins reunite the two hairpin ends to create a coding join, and the two blunt signal ends to create a signal join. The variation in this coding join creates the antigen receptor repertoire of the lymphocytes. Aberrant repair of the DNA DSBs could cause large deletions or translocations and lead to lymphoid tumors.

In order for V(D)J recombination to occur properly a cell must be kept in the G1 phase of the cell cycle. Ataxia-telangiectasia mutated (ATM) has been shown to have a function in the activation of check points of the cell cycle. After DNA DSBs are created, ATM activates protein p53, which prevents the cell from undergoing a G1 to S phase change. If the DNA DSBs are repaired, ATM activity decreases and p53 becomes inactive allowing the cell to change into the S phase. However, if the DNA DSBs are not repaired, the cell will remain in the G1 phase and undergo apoptosis.

ATM has also been shown to be involved in sensing the DSBs and amplifying the signal in order to initiate the repair of the DSBs. ATM senses the DSBs and amplifies the signal in order to recruit the NHEJ DSB repair proteins, which then repair the DSBs and complete V(D)J recombination.

In order to study these two mechanisms of ATM, we choose to look at the phosphorylation of downstream targets of ATM. To study the activation of checkpoints we chose p53 and Chk2, and to study the sensing and repair of DSBs we chose H2AX. It was found that the techniques used during this experiment did not provide the resolution necessary to determine if phosphorylation of the downstream targets of ATM occurred.