Synapsis of recombination signal sequences located in cis and DNA underwinding in V(D)J recombination

Molecular and Cellular Biology
Mihai Ciubotaru, David G Schatz

Abstract

V(D)J recombination requires binding and synapsis of a complementary (12/23) pair of recombination signal sequences (RSSs) by the RAG1 and RAG2 proteins, aided by a high-mobility group protein, HMG1 or HMG2. Double-strand DNA cleavage within this synaptic, or paired, complex is thought to involve DNA distortion or melting near the site of cleavage. Although V(D)J recombination normally occurs between RSSs located on the same DNA molecule (in cis), all previous studies that directly assessed RSS synapsis were performed with the two DNA substrates in trans. To overcome this limitation, we have developed a facilitated circularization assay using DNA substrates of reduced length to assess synapsis of RSSs in cis. We show that a 12/23 pair of RSSs is the preferred substrate for synapsis of cis RSSs and that the efficiency of pairing is dependent upon RAG1-RAG2 stoichiometry. Synapsis in cis occurs rapidly and is kinetically favored over synapsis of RSSs located in trans. This experimental system also allowed the generation of underwound DNA substrates containing pairs of RSSs in cis. Importantly, we found that the RAG proteins cleave such substrates substantially more efficiently than relaxed substrates and that underwinding may enh...Continue Reading

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Citations

Oct 29, 2011·Cell Death and Differentiation·M-L GougeonH Saïdi
Apr 2, 2005·Nature Reviews. Immunology·Michael T Lotze, Kevin J Tracey
Oct 2, 2007·Critical Care : the Official Journal of the Critical Care Forum·Mitchell P Fink
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Dec 20, 2007·The Journal of Biological Chemistry·Tadashi NishiharaHitoshi Sakano

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