DNA double strand breaks (DSBs) are perhaps the most harmful among all types of DNA damage because they involve physical separation of DNA molecules. If un-repaired or repaired aberrantly, DSBs can lead to loss (i.e. deletion) or abnormal rearrangement (i.e. translocation) of chromosomes, which can drive the genesis of a wide variety of cancers. DSBs can be generated by genotoxic stress, such as ionizing radiation, or as intermediates of normal cellular activities, such as the assembly of antigen receptor genes in lymphocytes that encode antibodies and T cell receptors that are key to fighting infection. During the assembly process, DNA DSBs are introduced at antigen receptor gene segments, and then repaired to generate the final DNA sequence encoding antigen receptor proteins. However, under certain circumstances, such DSBs cannot be properly repaired and eventually lead to the oncogenic deletions or translocations commonly seen in leukemias and lymphomas. Our laboratory previously identified a novel pathway that has an important role in protecting DSBs at antigen receptor genes from undergoing inappropriate processing by cellular enzymes and aberrant resolution. This pathway depends on the modification of a histone protein, H2AX, that is closely associated with cellular DNA. In response to DNA double strand breaks (DSBs), H2AX is phosphorylated by DNA damage sensor kinase ATM to form ?-H2AX that functions to recruit DNA damage response and repair proteins, and mediates additional modifications on other histone proteins. We have found that the phosphorylation of H2AX associated with broken DNA ends prevents processing of these DNA ends in ways that could lead to their aberrant repair. Here we propose to elucidate the mechanisms by which H2AX functions to prevent aberrant DSB repair. We hypothesize that ?-H2AX maintains DSB end structure through mechanisms that include modulating chromatin structure and DNA end accessibility through directing additional histone PTMs. In addition, given that ?-H2AX is shown to regulate protein association at DSBs, we predict that ?-H2AX can also impact the association of nucleases at DSB ends. To test our hypothesis, we will first reveal which histone modifications are generated in response to DSBs in a ?-H2AX-dependent manner and then determine whether ?-H2AX and ?-H2AX-dependent histone modifications modulate DNA end structures in a way that renders them less susceptible to degradation by nucleases. We will then determine whether ?-H2AX may prevent aberrant DNA end processing by limiting the association of nucleases with DSB ends. These studies will further our understanding of the genesis of chromosomal lesions that lead to lymphoid transformation and have broader implications for our understanding of the basis for genome instability in a variety of cancers.
DNA double strand breaks are generated during the assembly of lymphocyte antigen receptor genes. Aberrant repair of these DNA breaks causes oncogenic translocations and deletions that drive lymphoid tumorigenesis. We propose to study the mechanisms by which the DNA damage response factor and histone variant H2AX protects these DNA double strand breaks from being aberrantly processed and repaired to form transforming DNA lesions.
