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Abstract
Grant Number: 1R21GM071019-01 Project Title: Analyzing VDJ recombination at the single molecule level
PI Information: Name Title ROTH, DAVID B. roth@saturn.med.nyu.edu PROFESSOR Abstract: DESCRIPTION (provided by applicant): 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.
Public Health Relevance:
This Public Health Relevance is not available.Thesaurus Terms:
DNA, biophysics, gene rearrangement, nanotechnology, protein structure function
T cell receptor, cell differentiation, gene mutation, gene targeting, immunoglobulin gene, lymphocyte, molecular genetics
biotechnology, genetic technique, protein purification
Institution: NEW YORK UNIVERSITY SCHOOL OF MEDICINE 550 1ST AVE NEW YORK, NY 10016 Fiscal Year: 2004 Department: PATHOLOGY Project Start: 01-MAY-2004 Project End: 30-APR-2006 ICD: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES IRG: ALY