Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published April 7, 2015 | Supplemental Material + Published
Journal Article Open

Single-molecule analysis of RAG-mediated V(D)J DNA cleavage


The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. We have developed single-molecule assays to examine RSS binding by RAG1/2 and their cofactor high-mobility group-box protein 1 (HMGB1) as they proceed through the steps of this reaction. These assays allowed us to observe in real time the individual molecular events of RAG-mediated cleavage. As a result, we are able to measure the binding statistics (dwell times) and binding energies of the initial RAG binding events and characterize synapse formation at the single-molecule level, yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage on forming the synapse. Interestingly, we find that the synaptic complex has a mean lifetime of roughly 400 s and that its formation is readily reversible, with only ∼40% of observed synapses resulting in cleavage at consensus RSS binding sites.

Additional Information

Copyright © 2015 National Academy of Sciences. Freely available online through the PNAS open access option. Contributed by David Baltimore, February 20, 2015 (sent for review August 7, 2014; reviewed by L. James Maher) We thank members of the groups of D.G.S., D.B., and R.P. for helpful discussions and Caltech's Protein Expression Center. We also thank Michael Anaya, Jesse Bloom, Mihai Ciubotaru, Hernan Garcia, Jeff Gelles, Stephanie Johnson, Heun Jin Lee, Martin Linden, Jim Maher, Pradeep Ramesh, Philippe Rousseau, Laurence Salome, Patrick Swanson, and Catherine Tardin for discussions. We were supported by NIH Grants R37AI32524 (to D.G.S.), DP10D000217 (Director's Pioneer Award; to R.P.), and R01GM085286 (to R.P.); the Caltech–University of California, Los Angeles Joint Center for Translational Medicine (D.B.); the Sackler Foundation (D.B.); and La Fondation Pierre Gilles de Gennes (R.P.). Author contributions: G.A.L., R.C.B., D.G.S., D.B., and R.P. designed research; G.A.L. performed research; G.A.L., R.C.B., D.B., and R.P. contributed new reagents/analytic tools; G.A.L., R.C.B., D.G.S., D.B., and R.P. analyzed data; and G.A.L., R.C.B., D.G.S., D.B., and R.P. wrote the paper. Reviewers included: L.J.M., Mayo Clinic College of Medicine. The authors declare no conflict of interest. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1503477112/-/DCSupplemental.

Attached Files

Published - PNAS-2015-Lovely-E1715-23.pdf

Supplemental Material - pnas.201503477SI.pdf


Files (16.4 MB)
Name Size Download all
2.3 MB Preview Download
14.1 MB Preview Download

Additional details

August 22, 2023
October 20, 2023