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Flipping of alkylated DNA damage bridges base and nucleotide excision repair

Nature volume 459, pages 808–813 (2009)Cite this article

Abstract

Alkyltransferase-like proteins (ATLs) share functional motifs with the cancer chemotherapy target O6-alkylguanine-DNA alkyltransferase (AGT) and paradoxically protect cells from the biological effects of DNA alkylation damage, despite lacking the reactive cysteine and alkyltransferase activity of AGT. Here we determine Schizosaccharomyces pombe ATL structures without and with damaged DNA containing the endogenous lesion O6-methylguanine or cigarette-smoke-derived O6-4-(3-pyridyl)-4-oxobutylguanine. These results reveal non-enzymatic DNA nucleotide flipping plus increased DNA distortion and binding pocket size compared to AGT. Our analysis of lesion-binding site conservation identifies new ATLs in sea anemone and ancestral archaea, indicating that ATL interactions are ancestral to present-day repair pathways in all domains of life. Genetic connections to mammalian XPG (also known as ERCC5) and ERCC1 in S. pombe homologues Rad13 and Swi10 and biochemical interactions with Escherichia coli UvrA and UvrC combined with structural results reveal that ATLs sculpt alkylated DNA to create a genetic and structural intersection of base damage processing with nucleotide excision repair.

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Figure 1: Atl1 structure and lesion-binding site.
Figure 2: Atl1 DNA binding and damage sculpting.
Figure 3: Atl1 DNA lesion binding affinity and stoichiometry.
Figure 4: Biochemical and genetic connection of Atl1 to NER.
Figure 5: Alkyl-G lesion recognition allows NER repair of relatively non-distorting base lesions.

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Accession codes

Primary accessions

Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited in the Protein Data Bank under accession codes 3GVA (Atl1), 3GX4 (Atl1–O6-mG-DNA) and 3GYH (Atl1–O6-pobG-DNA).

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Acknowledgements

We thank C. C. Vu and J. Gong for aiding in the synthesis of O6-pobG oligomers, M. N. Boddy, J. Prudden and A. Sarker for performing genetics and biochemical experiments, G. Guenther, S. Pebernard, R. S. Williams, J. J. Perry, B. R. Chapados, M. Bjorås, D. S. Shin, K. Hitomi, C. Hitomi, E. D. Getzoff, G. Williams, S. Tsutakawa and P. K. Cooper for suggestions, and the staff at the Advanced Light Source (ALS) SIBYLS beamline and the Stanford Synchrotron Radiation Laboratory (SSRL). Operations at SSRL and ALS are supported by the US Department of Energy and NIH. This work was supported by National Institutes of Health grants CA097209 (J.A.T., A.E.P.), CA018137 (A.E.P.), GM070662 (M.G.F.), and CA59887 (L.A.P.), The Skaggs Institute for Chemical Biology (J.L.T.), North West Cancer Research Fund grant CR675 (O.F.), Cancer Research-UK (G.P.M.) and CHEMORES (G.P.M.).

Author Contributions M.D.K. and J.L.T. purified Atl1 protein and prepared Atl1–oligomer complexes for crystallization. A.S.A. and J.L.T. crystallized Atl1 and collected, processed and refined X-ray data. O6-pobG oligomers were synthesized by L.A.P. for crystallization and by C.M. and D.M.W. for surface plasmon resonance. A.J.W. and B.V. designed and synthesized oligonucleotides that contributed to the surface plasmon resonance data. A.M. and A.J.W. produced and characterized pure Atl1 protein for the surface plasmon resonance analyses. G.M. and M.T. performed surface plasmon resonance analyses. M.M. and M.G.F. performed electrophoretic mobility shift assays and analytical ultracentrifugation experiments and analysed the results. V.L. and A.B. generated Atl1 single and double deletants and Atl1-complement in S. pombe and carried out spot and clonogenic assays. R.K. and O.F. carried out the mutation assays in S. pombe. S.K. prepared constructs for and purified Atl1, E. coli Atl, N. vectensis ATL, AGT C145S, UvrA, UvrB and UvrC, and performed far western analyses, Atl1 expression assays in E. coli and ATL inhibition assays. M.F.S.-K. contributed intellectually to the initiation and design of the studies at the Paterson Institute. O.F., G.P.M., A.E.P. and J.A.T. provided intellectual guidance and research support. J.L.T. and J.A.T. wrote the paper. All authors discussed the results and manuscript.

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Author notes

  1. Rolf Kraehenbuehl, Oliver Fleck & Barbara Verbeek

    Present address: Present addresses: Cancer Research UK DNA Damage Response Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, UK (R.K.); Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark (O.F.); Department of Toxicology, University of Mainz, D-55131 Mainz, Germany (B.V.).,

Authors and Affiliations

  1. Skaggs Institute for Chemical Biology and Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA,

    Julie L. Tubbs, Andrew S. Arvai, Matthew D. Kroeger & John A. Tainer

  2. Cancer Research UK Carcinogenesis Group, Paterson Institute for Cancer Research, University of Manchester, Manchester, M20 4BX, UK ,

    Vitaly Latypov, Amna Butt, Andrew Marriott, Amanda J. Watson, Barbara Verbeek, Gail McGown, Mary Thorncroft & Geoffrey P. Margison

  3. Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA,

    Sreenivas Kanugula & Anthony E. Pegg

  4. Department of Molecular and Cellular Biochemistry, Center for Structural Biology, University of Kentucky, Lexington, Kentucky 40536, USA,

    Manana Melikishvili & Michael G. Fried

  5. NWCRF Institute, Bangor University, Gwynedd LL57 2UW, UK

    Rolf Kraehenbuehl & Oliver Fleck

  6. Institute of Human Genetics, Newcastle University, Newcastle-upon-Tyne, NE1 3BZ, UK ,

    Mauro F. Santibanez-Koref

  7. Department of Chemistry, Centre for Chemical Biology, University of Sheffield, Sheffield S3 7HF, UK

    Christopher Millington & David M. Williams

  8. Division of Environmental Health Sciences and the Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455, USA,

    Lisa A. Peterson

  9. Life Sciences Division, Bioenergy and Structural Biology, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA,

    John A. Tainer

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Correspondence to Anthony E. Pegg or John A. Tainer.

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This file contains Supplementary Methods and Data, Supplementary References, Supplementary Tables 1- 4 and Supplementary Figures 1-7 with Legends. Supplementary Table 1 was replaced on 18 June, 2009. (PDF 3602 kb)

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Tubbs, J., Latypov, V., Kanugula, S. et al. Flipping of alkylated DNA damage bridges base and nucleotide excision repair. Nature 459, 808–813 (2009). https://doi.org/10.1038/nature08076

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