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Published January 28, 2014 | public
Journal Article

Single Molecule Dynamics Governing the Initiation of V(D)J Recombination


The recombination activating genes (RAG)1 and RAG2 perform V(D)J recombination by rearranging conserved recombination signal sequences (RSSs) to generate antigen-receptors during lymphopoiesis. However the orchestration of V(D)J recombination on biologically relevant (long) length scales has resisted experimental investigation. Here we develop single-molecule assays to watch in real time as RAG1/2 and its co-factor HMGB1 carry out V(D)J recombination from start (RSS binding) to finish (hairpin formation) on long DNA molecules. We capture various intermediate states preceding hairpin formation, show how RAG1/2 and HMGB1 form bends on the DNA, demonstrate how the identity of the recombination signal sequence modulates bending with single bp resolution and show HMGB1 must compact DNA flanking RSSs to form hairpins. Our results provide single-molecule mechanistic insight into the orchestration of V(D)J recombination.

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© 2014 Biophysical Society. Published by Elsevier Inc.

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