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Amino acid residues in Rag1 crucial for DNA hairpin formation

Nature Structural & Molecular Biology volume 13, pages 1010–1015 (2006)Cite this article

Abstract

The Rag proteins carry out V(D)J recombination through a process mechanistically similar to cut-and-paste transposition. Specifically, Rag complexes form DNA hairpins through direct transesterification, using a catalytic Asp-Asp-Glu (DDE) triad in Rag1. How is sufficient DNA distortion introduced to allow hairpin formation? We hypothesized that, like certain transposases, the Rag proteins might use aromatic amino acid residues to stabilize a flipped-out base. Through in vivo and in vitro experiments and structural predictions, we identified residues in Rag1 crucial for hairpin formation. One of these, a conserved tryptophan (Trp893), probably participates in base-stacking interactions near the cleavage site, as do Trp298, Trp265 and Trp319 in the Tn5, Tn10 and Hermes transposases, respectively. Other residues surrounding the catalytic glutamate (YKEFRK) may share functional similarities with the YREK motif in IS4 family transposases.

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Figure 1: An unbiased search for residues involved in hairpin formation.
Figure 2: Analysis of cleavage reactions on matched or mismatched (MM) oligonucleotide substrates.
Figure 3: Weak hairpin activity of W893A and Y935A stimulated by synapsis.
Figure 4: Structure of Hermes transposase, highlighting potential positions of mechanistically important residues in Rag1.

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Acknowledgements

The authors thank members of the Roth laboratory for thoughtful discussions and comments on the manuscript and G. Weller for unpublished data. This work was supported by US National Institutes of Health grant AI36420 and funding from the Irene Diamond Foundation (to D.B.R.).

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Authors and Affiliations

  1. Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine, New York, 10016, New York, USA

    Catherine P Lu, Vicky L Brandt & David B Roth

  2. Department of Pathology, New York University School of Medicine, New York, 10016, New York, USA

    Catherine P Lu, Vicky L Brandt & David B Roth

  3. Department of Immunology, Baylor College of Medicine, Houston, 77030, Texas, USA

    Hector Sandoval

  4. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, 60637, Illinois, USA

    Phoebe A Rice

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  1. Catherine P Lu
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  2. Hector Sandoval
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  4. Phoebe A Rice
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  5. David B Roth
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Contributions

H.S. began the mutagenesis project while a student in the Roth lab at Baylor College of Medicine. C.P.L. performed all the experiments shown; P.A.R. performed the structural predictions and analysis; C.P.L., V.L.B. and D.B.R. thought about experiments, analyzed data and wrote the manuscript.

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Correspondence to David B Roth.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Nicking and hairpin formation by Rag1/Rag2. (PDF 182 kb)

Supplementary Fig. 2

Time-course analysis and prenicked substrate experiments. (PDF 500 kb)

Supplementary Fig. 3

Comparative alignment of Tn5, Tn10 and Rag1. (PDF 184 kb)

Supplementary Fig. 4

Rough alignment of proposed catalytic domains of Hermes and Rag1. (PDF 79 kb)

Supplementary Table 1

Oligonucleotide sequences. (PDF 29 kb)

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Lu, C., Sandoval, H., Brandt, V. et al. Amino acid residues in Rag1 crucial for DNA hairpin formation. Nat Struct Mol Biol 13, 1010–1015 (2006). https://doi.org/10.1038/nsmb1154

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