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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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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41586_2000_BF35000249_MOESM1_ESM.gif
Figure 1: DNA-bound AP Endonuclease (APE1) structures and mutants reveal abasic DNA binding to coordinate DNA repair (animated version) (GIF 954 kb)
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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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DOI: https://doi.org/10.1038/35000249
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