The human V(D)J recombinase mistakenly catalyzes chromosomal translocations in almost 50% of human lymphoid malignancies. In the first five years of human life, such malignancies account for half of all cancers. The V(D)J recombination reaction normally assembles the variable domain exon of antigen receptors in lymphoid cells, and this is a critical step for the development of the entire immune system. The latter half of this reaction shares essentially identical features and probably enzymatic components with general DNA end joining (double-strand break repair). Hence, knowledge of the V(D)J recombination reaction will be important for cancer etiology generally. The first assay for studying the V(D)J recombination reaction and the recombinase in human cells was developed under the support of this grant during the previous funding period. Based on this assay, imbalances of products were identified in the human V(D)J recombination reaction in two human acute lymphoblastic leukemia cell lines. Such imbalances are not observed in murine lymphoid cells when studied with the identical V(D)J recombination substrate region. These imbalances are the very feature that may account for the frequency of chromosomal translocations in human lymphoid malignancies.
The aims of the current proposal are as follows. First, the extent of the V(D)J recombination reaction product imbalance will be examined in neoplastic human lymphoid cells and compared to cells from normal individuals. Physical methods of detection will also be employed in this comparison. Factors that correlate with or affect this imbalance of reaction products will also be explored. Second, components of the human V(D)J recombinase will be examined or identified that account for abnormalities of the human V(D)J recombination reaction. This will involve examination of already identified components as well as the isolation of yet-unidentified components. Third, the human assay will be applied to understanding the recombinagenic nature of major chromosomal breakpoint sites that account for a large fraction of events in human lymphoid cancer. In the fourth aim, the issue of genomic accessibility in human cells will be explored. Most of the genome is not accessible to the V(D)J recombinase or other enzymes that cause double-strand breaks. In the previous funding period, we determined that one major component that determines accessibility is CpG methylation. The maintenance of the inaccessible state is critical to maintaining genome stability generally, but especially during periods of expression of V(D)J recombinase activity. Here the ability of neoplastic human lymphoid cells to maintain this inaccessible state will be compared with that in the corresponding cells from normal individuals. Given that we have found important differences between the murine and human recombinases, it is critical that the reaction and recombinase enzyme be studied in normal and neoplastic human cells to fully define the features and steps that result in chromosomal translocations.
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