TdT and pol mu in DNA repair and immune system diversity

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DESCRIPTION (provided by applicant): The mammalian non-homologous end joining (NHEJ, end joining) pathway is employed in all cell types to repair DNA double strand breaks (DSBs) caused by DNA damaging agents (e.g. ionizing radiation, chemotherapeutic drugs). End joining is also essential for efficient resolution of DSB intermediates during V(D)J recombination, a lymphoid specific process required to assemble the immune system's antigen specific receptors. Defective end joining thus results in radiosensitivity, an increased incidence of cancer, as well as immunodeficiency. Two related DNA polymerases, the lymphoid-specific Terminal deoxynucleotidyl transferase (TdT) and the recently described polymerase mu (pol mu), specifically associate with factors required for end joining. 1) The role of pol mu in end joining is as yet unclear. Cellular V(D)J recombination assays will be used to clarify pol mu's role in V(D)J recombination, as well as its role in end joining repair in general. 2) Previous cellular experiments have outlined characteristic activities of polymerases in end joining, such that this pathway is more accurate than would be expected otherwise. A reduced system, using purified factors, will be used to determine if pol mu, TdT, or other polymerases possess these characteristic activities. Target mutations in pol mu or TdT will also be made to better understand the importance of the ability of these specific polymerases to associate with end joining factors in achieving more accurate end joining. 3) TdT and pol mu readily incorporate both DNA and RNA during synthesis in vitro, while all other known nucleic acid polymerases (RNA or DNA) typically incorporate the appropriate nucleic acid type at least 1000 times more efficiently than the inappropriate nucleic acid type. Experiments will be performed do determine if this activity can also be observed at sites of V(D)J recombination (or end joining DSB repair) in cells, and what impact RNA incorporation by these polymerases has on end joining in vitro. This work will provide a comprehensive understanding how polymerases in specific, and end processing factors in general, are employed by the end joining pathway for DSB repair such that this repair pathways is accurate. It will help address how the activity of these processing factors are controlled, how end processing is achieved with acceptable risk, and what the consequences of un-controlled processing activity might be to genome stability.