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Amino acid residues in Rag1 crucial for DNA hairpin formation

Nature Structural & Molecular Biology volume 13, pages 10101015 (2006) | Download Citation

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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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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thank members of the Roth laboratory for thoughtful discussions and comments on the manuscript and G. Weller for unpublished data.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.).

Author information

Affiliations

  1. Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine, and Department of Pathology, New York University School of Medicine, New York, New York 10016, USA.

    • Catherine P Lu
    • , Vicky L Brandt
    •  & David B Roth
  2. Department of Immunology, Baylor College of Medicine, Houston, Texas 77030, USA.

    • Hector Sandoval
  3. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA.

    • Phoebe A Rice

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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to David B Roth.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Nicking and hairpin formation by Rag1/Rag2.

  2. 2.

    Supplementary Fig. 2

    Time-course analysis and prenicked substrate experiments.

  3. 3.

    Supplementary Fig. 3

    Comparative alignment of Tn5, Tn10 and Rag1.

  4. 4.

    Supplementary Fig. 4

    Rough alignment of proposed catalytic domains of Hermes and Rag1.

  5. 5.

    Supplementary Table 1

    Oligonucleotide sequences.

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DOI

https://doi.org/10.1038/nsmb1154

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