The long-term objective of this research is to understand the influence of hematopoietic-specific developmental programs on the repair of one type of DNA damage--a double strand break (DSB)--and the initial molecular events that lead to translocations, which are a hallmark of leukemia, lymphoma, and soft-tissue sarcomas. Chromosomal DSBs are formed during normal metabolic processes including DNA replication and antigen receptor rearrangement in developing lymphoid cells, as well as following exposure to DNA damaging agents. There is an association between certain chemotherapeutic agents and secondary leukemia, e.g. treatment with topoisomerase II inhibitors and rearrangements involving 1lq23, and sequence analysis of breakpoints suggests that DNA damage is involved in the translocation process. However, the mechanisms by which specific translocations occur within developing hematopoietic subpopulations in the initial formation of rearrangements remain unclear. These questions can be addressed by adaptation of a genetic system developed in murine ES cells based on the rare-cutting yeast endonuclease I-Sce I to introduce DSBs at defined genomic loci and analyze recombinant repair events at the molecular level. This system also scores for translocations, duplications, and deletions that may result. This proposal will adapt the I-Sce I system to examine DSB repair and recombination in hematopoietic early progenitor and myeloid cell lineages and the potential for this type of damage to promote illegitimate recombination. Approaches will: (1) determine the potential for repair of DSBs and interchromosomal recombination to promote genome rearrangements within specific lineages; (2) determine the potential for repair of DSBs within the breakpoint cluster regions of the MLL and AF-4 genes to result in t(4;11) translocations similar to those observed in the clinical setting; and (3) use a genomics microarray-based approach to characterize the influence of the stage of hematopoietic development or the damaging agent on the specificity of a cell's DNA damage response. These studies will provide important insight into the biology of DSB rejoining in hematopoietic cell subpopulations, and the factors responsible for the normal suppression of genome rearrangements and, ultimately, tumorigenesis. Unraveling the etiology and consequences of translocations may lead to new approaches to therapy and prevention.
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