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Albany 2015:Book of Abstracts

Albany 2015
Conversation 19
June 9-13 2015
©Adenine Press (2012)

V(D)J recombination targeted in cis by transcription induced DNA supercoiling

Somatic recombination assembles and diversifies the antigen receptor genes, of B and T cells. RAG recombinase binds two specific DNA sites (RSSs) to initiate recombination, a process called paired complex (PC) or synapsis formation. Synapsis is followed by cleavage and then by repair of the double stranded breaks which physiologically occur only between gene segments located intrachromosomally. Pairing preserves the integrity of the genome, preventing aberrant interchromosomal joinings (translocations) implicated in human B and T cell lymphomas. More than half of all childhood cancers derive from lymphoid cells and most of these malignancies are due to aberrant recombination translocations. Despite its significance the mechanism that physiologically directs somatic recombination for sites located in cis with respect to DNA is entirely unknown. The central hypothesis, suggested by our data, sets DNA supercoiling as a major dynamic feature controlling recombination between sites located on the same chromosome. Prior to recombination, dechromatinization, and germline transcription of the variable gene segments of the immunoglobulin (Ig) or T cell receptor (TCR) loci leads to a substantial dynamic change in the torsional tension and supercoiling of the adjacent DNA. We study the role of DNA supercoiling as an active factor in controlling RAG intrachromosomal sites pairing. Using DNA circles containing RSS sites of defined topology, we study how the rate and efficiency of RAG synapsis is affected by their topological configuration. To supercoil the DNA subjected to recombination and to test in vivo the targeting mechanism we use a heterologous controllable transcription unit in the proximity of reporter RSSs. Our study offers a new conceptual understanding of the recombinase gene accessibility via a topological filter mechanism. Thus, our work provides the ground for testing and understanding how aberrant pairing may lead to translocations, with major implications in human disease.

Mihai Ciubotaru 1,2

1Department of Immunobiology
2Department of Molecular Biophysics and Biochemistry,
Yale University School of Medicine
New Haven, CT 06511, USA
2National Institute for Physics and Nuclear Engineering Horia Hulubei
Department of Life and Environmental Physics
Atomistilor Str., 077125,
Bucharest-Magurele, Romania

Phone: 001-40-740027747
mihai.ciubotaru@yale.edu