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Intron-encoded homing endonuclease I-TevI also functions as a transcriptional autorepressor

Nature Structural & Molecular Biology volume 11, pages 936–944 (2004)Cite this article

Abstract

Customary binding sites of intron-encoded homing endonucleases lie within cognate intronless alleles, at the so-called homing sites. Here, we describe a novel, high-affinity binding site for I-TevI endonuclease, encoded within the group I td intron of phage T4. This site is an operator that overlaps the T4 late promoter, which drives I-TevI expression from within the td intron. I-TevI binds the operator and homing sites with equal affinity, and functions as a transcriptional autorepressor. Distinct sequence and spacing requirements of the catalytic domain result in reduced cleavage activity on operator DNA. Crystallographic studies showed that the overall interactions of the DNA-binding domain with the operator and homing sites are similar, but have some different hydrogen-bonding contacts. We present a model in which the flexibility in protein-DNA interactions allows I-TevI to bind variant intronless alleles to promote intron mobility while facilitating its function in autorepression, and thereby persistence in its host.

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Figure 1: Regulatory sites for intron endonucleases in T-even phages.
Figure 2: I-TevI 130C binds with equal affinity to the homing and to the operator sites.
Figure 3: Interactions of I-TevI with the operator site.
Figure 4: I-TevI cleavage efficiency is reduced on operator substrate.
Figure 5: In vivo activity of the I-TevI promoter in the presence and absence of I-TevI.
Figure 6: Cocrystal structure of I-TevI 130C on operator or homing site DNA.
Figure 7: Comparison of alternative minor groove hydrogen-bonding contacts.
Figure 8: Distinct functional domains allow I-TevI to act as an endonuclease and a repressor.

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Acknowledgements

We thank L. Conlan, J. Dansereau, and J. Pata for their thoughtful comments on the manuscript, J. Dansereau for figure refinements and M. Carl for expert manuscript preparation. This work was supported by US National Institutes of Health (NIH) grants GM39422 and GM44844 to M.B., GM56966 to P.V.R. and GM37746 to D.A.S. D.R.E. was supported by a postdoctoral fellowship from the Canadian Institute of Health Research. S.L. was supported in part by a stipend from the Center for Molecular Genetics, University at Albany. The X-ray diffraction facilities at beamline X12C of the National Synchrotron Light Source were supported by the US Department of Energy and by grants from the NIH. Oligonucleotides were provided by the Molecular Genetics Core Facility of Wadsworth Center.

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Author notes

  1. David R Edgell

    Present address: Department of Biochemistry, Medical Sciences Building, University of Western Ontario, London, Ontario, N6A 5C1, Canada

  2. Stephen LaBonne

    Present address: Lake County Crime Laboratory, 235 Fairgrounds Road, Painesville, Ohio, 44077, USA

  3. Thomas M Boyd

    Present address: Department of Emergency Medicine, Jacobi Medical Center, 1400 Pelham Parkway, Bronx, New York, 10461, USA

Authors and Affiliations

  1. New York State Department of Health, Wadsworth Center, Center for Medical Sciences, 150 New Scotland Avenue, Albany, 12208, New York, USA

    David R Edgell, Victoria Derbyshire, Patrick Van Roey, Matthew J Stanger, Zhong Li & Marlene Belfort

  2. Department of Biological Sciences, University at Albany-SUNY, 1400 Washington Avenue, Albany, 12222, New York, USA

    Stephen LaBonne, Thomas M Boyd & David A Shub

  3. Department of Biochemistry, Medical Sciences Building, University of Western Ontario, London, Ontario, N6A 5C1, Canada

    Stephen LaBonne & Thomas M Boyd

  4. Lake County Crime Laboratory, 235 Fairgrounds Road, Painesville, Ohio, 44077, USA

    David R Edgell & Thomas M Boyd

  5. Department of Emergency Medicine, Jacobi Medical Center, 1400 Pelham Parkway, Bronx, New York, 10461, USA

    David R Edgell & Stephen LaBonne

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Correspondence to Marlene Belfort.

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Edgell, D., Derbyshire, V., Roey, P. et al. Intron-encoded homing endonuclease I-TevI also functions as a transcriptional autorepressor. Nat Struct Mol Biol 11, 936–944 (2004). https://doi.org/10.1038/nsmb823

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