The RAG1 Homeodomain Recruits HMG1 and HMG2 To Facilitate Recombination Signal Sequence Binding and To Enhance the Intrinsic DNA-Bending Activity of RAG1-RAG2

Abstract

V(D)J recombination is initiated by the specific binding of the RAG1-RAG2 (RAG1/2) complex to the heptamer-nonamer recombination signal sequences (RSS). Several steps of the V(D)J recombination reaction can be reconstituted in vitro with only RAG1/2 plus the high-mobility-group protein HMG1 or HMG2. Here we show that the RAG1 homeodomain directly interacts with both HMG boxes of HMG1 and HMG2 (HMG1,2). This interaction facilitates the binding of RAG1/2 to the RSS, mainly by promoting high-affinity binding to the nonamer motif. Using circular-permutation assays, we found that the RAG1/2 complex bends the RSS DNA between the heptamer and nonamer motifs. HMG1,2 significantly enhance the binding and bending of the 23RSS but are not essential for the formation of a bent DNA intermediate on the 12RSS. A transient increase of HMG1,2 concentration in transfected cells increases the production of the final V(D)J recombinants in vivo.

ACKNOWLEDGMENTS

We are grateful to H. Clevers and M. van de Wetering for the TCF-1b cDNA clones, D. Schatz and M. Difilippantonio for the VP16-RAG and luciferase plasmids, D. Thanos for the HMG-I(Y) cDNA clone and pBend2 vectors, T. Wirth for the HMG2 cDNA, and A. Hodtsev and A. Han for critical reading of the manuscript.

This work was supported by NIH grant AI40191 to E.S. and AIRC and MURST grants to M.E.B. E.S. was a Cancer Research Institute Clinical Investigator and Howard Hughes Medical Institute Assistant Investigator.