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DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination

Nature volume 403, pages 451–456 (2000)Cite this article

An Erratum to this article was published on 30 March 2000

Abstract

Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases1. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5′ of AP sites to prime DNA repair synthesis2,3,4. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized β-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.

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Figure 1: Stereo views of simulated-annealed omit electron density for APE1–DNA complexes showing the flipped-out abasic site and phosphodiester bond cleavage.
Figure 2: Stereo views of the APE1 complex with AP-DNA showing double-loop penetration of the DNA helix, plus charge and surface complementarity.
Figure 3: APE1 interactions with the flipped-out AP site provide damage specificity and suggest a specific reaction mechanism for phosphodiester bond cleavage.
Figure 4: Activities and kinetics of wild-type and mutant human APE1 enzymes.

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Acknowledgements

We thank S. S. Parikh, C. D. Putnam, D. S. Daniels and D. J. Hosfield for helpful discussions, and the staff and facilities at SSRL. This work was supported by the NIH, by a Laboratory Directed Research and Development grant from the Lawrence Berkeley Laboratory, by a cancer research supplement administered through LBNL from the National Cancer Institute (to J.A.T., S.M. and P. Cooper), the Skaggs Institute for Chemical Biology and a Special Fellowship from the Leukemia Society of America (to C.D.M.).

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  1. Skaggs Institute for Chemical Biology and the Department of Molecular Biology, MB-4, The Scripps Research Institute, 10550 North Torrey Pines Rd., La Jolla, 92037-1027, California, USA

    Clifford D. Mol & John A. Tainer

  2. Sealy Center for Molecular Science Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, 77555-1079 , Texas, USA

    Tadahide Izumi & Sankar Mitra

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  1. Clifford D. Mol
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Correspondence to John A. Tainer.

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Mol, C., Izumi, T., Mitra, S. et al. DNA-bound structures and mutants reveal abasic DNA binding by APE1 DNA repair and coordination. Nature 403, 451–456 (2000). https://doi.org/10.1038/35000249

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