Letter | Published:

The DNA glycosylase AlkD uses a non-base-flipping mechanism to excise bulky lesions

Nature volume 527, pages 254258 (12 November 2015) | Download Citation

Abstract

Threats to genomic integrity arising from DNA damage are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases1,2. How these enzymes find small modifications within the genome is a current area of intensive research. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket2,3,4,5. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. Here we present the first, to our knowledge, DNA glycosylase mechanism that does not require base flipping for either binding or catalysis. Using the DNA glycosylase AlkD from Bacillus cereus, we crystallographically monitored excision of an alkylpurine substrate as a function of time, and reconstructed the steps along the reaction coordinate through structures representing substrate, intermediate and product complexes. Instead of directly interacting with the damaged nucleobase, AlkD recognizes aberrant base pairs through interactions with the phosphoribose backbone, while the lesion remains stacked in the DNA duplex. Quantum mechanical calculations revealed that these contacts include catalytic charge–dipole and CH–π interactions that preferentially stabilize the transition state. We show in vitro and in vivo how this unique means of recognition and catalysis enables AlkD to repair large adducts formed by yatakemycin, a member of the duocarmycin family of antimicrobial natural products exploited in bacterial warfare and chemotherapeutic trials6,7. Bulky adducts of this or any type are not excised by DNA glycosylases that use a traditional base-flipping mechanism5. Hence, these findings represent a new model for DNA repair and provide insights into catalysis of base excision.

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Accessions

Data deposits

Atomic coordinates and structure factors have been deposited in the Protein Data Bank under accession codes 5CL3, 5CL4, 5CL5, 5CL6, 5CL7, 5CL8, 5CL9, 5CLA, 5CLB, 5CLC, 5CLD and 5CLE.

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Acknowledgements

We thank C. Rizzo and T. Johnson Salyard for assistance with oligodeoxynucleotide synthesis and E. Skaar and L. Mike for assistance with genetic manipulation of B. anthracis. This work was funded by the National Science Foundation (MCB-1122098 and MCB-1517695 to B.F.E.) and the National Institutes of Health (R01ES019625 to B.F.E. and R01CA067985 to S.S.D.). Support for the Vanderbilt Robotic Crystallization Facility was also provided by the National Institutes of Health (S10RR026915). Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy Office of Science by Argonne National Laboratory, was supported by the US Department of Energy (DE-AC02-06CH11357). Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (085P1000817). E.A.M. and Z.D.P. were partially supported by the Vanderbilt Training Program in Environmental Toxicology (T32ES07028).

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Affiliations

  1. Department of Biological Sciences and Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232, USA

    • Elwood A. Mullins
    • , Rongxin Shi
    • , Zachary D. Parsons
    •  & Brandt F. Eichman
  2. Department of Chemistry, University of California, Davis, California 95616, USA

    • Philip K. Yuen
    •  & Sheila S. David
  3. Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan

    • Yasuhiro Igarashi

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Contributions

E.A.M. and B.F.E. conceived the project; E.A.M., R.S., Z.D.P. and B.F.E. designed experiments; E.A.M. performed biochemical and structural experiments; R.S. performed cellular experiments; Z.D.P. performed computational experiments; P.K.Y., S.S.D. and Y.I. supplied reagents; E.A.M., R.S., Z.D.P. and B.F.E. analysed data and wrote the paper; all authors commented on the manuscript.

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The authors declare no competing financial interests.

Corresponding author

Correspondence to Brandt F. Eichman.

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https://doi.org/10.1038/nature15728

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