DNA Double strand breaks (DSBs) in chromosomes can be caused by exogenous damaging agents (e.g. ionizing radiation), but are also produced as intermediates in normal recombination events, including V (D) J recombination. DSBs are lethal, thus must be repaired for cells to live; aberrant DSB repair is also linked to frequent tumor development. End joining in mammals requires at least Ku, DNA-PKcs, XRCC4, DNA Ligase IV, and Artemis. However, how these factors function in these processes at the molecular level is not well understood, and even less is known how their function is impacted by chromatin structure. I. What is the function of DNA-PK kinase activity? The target of DNA-PKcs kinase activity most relevant in an in vitro end-joining assay will be determined. The hypothesis that kinase activity is a critical factor in ensuring DSBs are protected from modification until they are ready to be repaired will also be tested. II. How is nuclease activity accurately employed by end joining? Artemis is a recently identified nuclease that has a clear role in V (D) J recombination, but a more ambiguous role in general end joining repair. In vitro experiments will be performed to cladfy how other end joining factors impact Artemis activity, and if Artemis activity is controlled in an in vitro end joining reaction in an appropriate, context-dependent manner. III. How does chromatin structure impact targeting and repair of DSBs in V (D) J recombination? Covalent modification of histones directs alterations in chromatin structure. These alterations in chromatin structure in turn are cdtical for the developmentally specific targeting of DSBs in V (D) J recombination, and they also play an important role in general repair of DSBs by the end joining pathway. A cell line model will be employed to determine what modifications are associated with inductien and resolution of DSB intermediates in V (D) J recombination. The consequences of preturbing-selected modifications will also be assessed. IV. How is the function of DSB repair factors effected by the nucleosome? Purified, defined mononucleosomes will be used to determine how this fundamental unit of chromatin structure alters the activities of core end joining components (Ku, DNA-PKcs, XRCC, 4, ligase IV) in vitro. Together, these aims will clarify how end joining works at the molecular level, and provide insight into how aberrant rearrangements occur when V (D) J recombination and end joining DSB repair goes awry.
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