The end-joining pathway has an important role in all cell types repairing DSBs caused by certain DNA damaging agents (e.g. ionizing radiation). End joining is also essential for efficient resolution of DSB intermediates during V(D)J recombination, a lymphocyte specific process required for assembly of the immune system's antigen specific receptors. Defective end joining thus results in radiation sensitivity, an increased incidence of cancer, and severe immunodeficiency. My lab uses diverse molecular biological approaches to develop an understanding how end joining works, and what happens when it doesnt. Previous in vitro characterizations of the end joining pathway have used "naked DNA" as a substrate - we are interested in now learning how the mechanism adapts to more biologically relevant "chromatinized" substrates. Do nucleosomes present a barrier to this pathway, as it does with other processes? For example, the Ku heterodimer is a core factor within this pathway that loads on free DNA-ends, then translocates inward. Broken ends closely associated with a nucleosome surface (how close?) would likely disrupt loading of Ku, and would almost certainly block inward translocation. Are there specific enzymes that help remodel the nucleosome for end joining? Which ones, and how? A graduate student in my lab is interested in determining the role pol mu, a recently described mammalian polymerase, has in end joining DSB repair. While pursuing these experiments, she determined that pol mu surprisingly incorporates both RNA and DNA during repair. This contradicts a basic tenet of molecular biology: all other DNA polymerases have so far proven to be highly specific, typically incorporating the "appropriate" nucleic acid 3-4 orders of magnitude more efficiently than the "inappropriate" nucleic acid . We therefore propose to deterimine if pol mu in fact incorporates RNA into mammalian genomes in cells, and what the biological role of this activity might be.