RAG Transposase Can Capture and Commit to Target DNA before or after Donor Cleavage

Abstract

The discovery that the V(D)J recombinase functions as a transposase in vitro suggests that transposition by this system might be a potent source of genomic instability. To gain insight into the mechanisms that regulate transposition, we investigated a phenomenon termed target commitment that reflects a functional association between the RAG transposase and the target DNA. We found that the V(D)J recombinase is quite promiscuous, forming productive complexes with target DNA both before and after donor cleavage, and our data indicate that the rate-limiting step for transposition occurs after target capture. Formation of stable target capture complexes depends upon the presence of active-site metal binding residues (the DDE motif), suggesting that active-site amino acids in RAG-1 are critical for target capture. The ability of the RAG transposase to commit to target prior to cleavage may result in a preference for transposition into nearby targets, such as immunoglobulin and T-cell receptor loci. This could bias transposition toward relatively “safe” regions of the genome. A preference for localized transposition may also have influenced the evolution of the antigen receptor loci.

ACKNOWLEDGMENTS

We thank Tania Baker and Ilana Goldhaber-Gordon for many helpful discussions; Vicky Brandt for editorial assistance; Meni Melek for providing physical assay conditions; Ilana Goldhaber-Gordon, S. Kale, J. Qiu, L. E. Huye, and H. Yarnall-Schultz for valuable comments on the manuscript; and L. E. Huye for HMG1 protein. S. Robertson and D. Guzman provided secretarial assistance. We also thank anonymous referees for insightful suggestions.

D.B.R. is an Assistant Investigator of the Howard Hughes Medical Institute. M.A.L. was supported by a predoctoral fellowship from the National Institutes of Health (T32-AI07495). This work was supported by a grant from the National Institutes of Health (AI-36420).