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Two-metal active site binding of a Tn5 transposase synaptic complex

Nature Structural Biology volume 9, pages 278–281 (2002)Cite this article

Abstract

A synaptic complex of Tn5 transposase with an extended outside end DNA duplex was prepared and crystallized, and its crystal structure was determined in an effort to reveal the role of metal ions in catalysis. Two Mn2+ ions bound to the active site when a single nucleotide of donor DNA was added to the 3′ end of the transferred strand. Marked conformational changes were observed in the DNA bases closest to the active site. The position of the metal ions and the conformational changes of the DNA provide insight into the mechanism of hairpin formation and cleavage, and is consistent with a two-metal model for catalysis.

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Figure 1: Proposed mechanism for donor DNA cleavage and hairpin formation/resolution13.
Figure 2: Active site view of Tn5–Mn2.
Figure 3: Comparison of the DNA structure of Tn5–Mn2 (purple) and Tn5–Mn (gold).
Figure 4: The salt bridge formed between Glu 110 and Lys 345 for Tn5–Mn2 is depicted in green.

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Acknowledgements

We thank N. Duke and A. Joachimak at the Advanced Photon Source SBC beamline for help with data collection. The work was supported in part by a grant from the NIH. The use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Energy Research We thank the reviewers for their insightful comments.

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  1. Department of Biochemistry, University of Wisconsin, Madison, 53706, Wisconsin, USA

    Scott Lovell, Igor Y. Goryshin, William R. Reznikoff & Ivan Rayment

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  1. Scott Lovell
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  2. Igor Y. Goryshin
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Correspondence to Ivan Rayment.

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Lovell, S., Goryshin, I., Reznikoff, W. et al. Two-metal active site binding of a Tn5 transposase synaptic complex. Nat Struct Mol Biol 9, 278–281 (2002). https://doi.org/10.1038/nsb778

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