V(D)J recombination, a site-specific DNA rearrangement reaction, is responsible for assembling immunoglobulin and T cell receptor variable region genes during lymphocyte differentiation, and it is essential for the generation of B and T lymphocytes. Aberrant V(D)J recombination events underlie a considerable fraction of lymphoid neoplasms, which are among the most common cancers. The actions of a single molecule are of utmost importance in these reactions, yet they elude the approaches available to us through """"""""bulk biochemistry."""""""" No one has yet applied single-molecule studies to site-specific recombination; the studies we propose herein will be the first such studies conducted to understand DNA rearrangement using single-DNA micromanipulation techniques. Two specific DNA-protein complexes perform key regulatory functions in the V(D)J recombination reaction. The synaptic complex, comprising the RAG-1 and RAG-2 proteins plus accessory molecules, brings together the two DNA segments that are to undergo recombination. After DNA cleavage, the RAG proteins remain associated with the broken DNA ends in the form of a post-cleavage complex that helps direct proper joining. These two RAG-DNA complexes are critical to safeguarding the genome, yet we know little about their formation or specific activities. We propose to develop new biophysical tools to directly observe and control their formation. Specifically, we will 1) construct the necessary DNA substrates and a molecular tweezer apparatus; 2) analyze V(D)J synaptic complex formation on single tethered DNA molecules; and 3) analyze the post-cleavage complex using single-DNA studies. ? ? ? ?
| Adams, Christian D; Schnurr, Bernhard; Marko, John F et al. (2007) Pulling apart catalytically active Tn5 synaptic complexes using magnetic tweezers. J Mol Biol 367:319-27 |
| Skoko, Dunja; Yoo, Daniel; Bai, Hua et al. (2006) Mechanism of chromosome compaction and looping by the Escherichia coli nucleoid protein Fis. J Mol Biol 364:777-98 |
| Adams, Christian D; Schnurr, Bernhard; Skoko, Dunja et al. (2006) Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences. Mol Microbiol 62:1558-68 |
