V(D)J recombination is responsible for mistaken chromosomal translocations in almost 50% of human lymphoid malignancies. The goal of this proposal is to determine the molecular basis for how these chromosomal translocations occur. Three specific hypotheses for such chromosomal translocations are proposed, and experimental strategies are described to test these proposed mechanisms. The first hypothesis (Aim 1) proposes misrecognition of cryptic heptamer/nonamer signal sequences. This hypothesis can be tested by utilizing the human V(D)J recombination assay developed by the investigator. The major breakpoint regions for bcl-2, SCL, SIL, Hox11, Ttg-1, and LMO2 will be quantitatively tested for their recombination activity. The effect of transcription through these regions and the effect of the surrounding DNA sequences will be tested. The second hypothesis (Aim 2) proposes that signal end intermediates of V(D)J recombination function as transposons and catalyze chromosomal translocations. This mechanism will be tested in lymphoid cells from normal individuals and from cells derived from lymphoid malignancies. This mechanism could account for reciprocal translocations or in isolated chromosome breaks that are then paired with V, D, or J segments. The third hypothesis (Aim 3) proposes that some major breakpoint regions are prone to breakage not because they are misrecognized for cutting by the V(D)J recombination enzymes, but rather because they are fragile and subject to nuclease action (breakage) because they assume a non-B DNA conformation. The investigator proposes to examine the structure of DNA sequences at translocation breakpoints. Corresponding studies of such regions on minichromosomes will permit the testing of the effects of the direction of DNA replication and transcription.
Showing the most recent 10 out of 14 publications
