Escherichia coli AlkB and its human homologues ABH2 and ABH3 repair DNA/RNA base lesions by using a direct oxidative dealkylation mechanism. ABH2 has the primary role of guarding mammalian genomes against 1-meA damage by repairing this lesion in double-stranded DNA (dsDNA), whereas AlkB and ABH3 preferentially repair single-stranded DNA (ssDNA) lesions and can repair damaged bases in RNA. Here we show the first crystal structures of AlkB–dsDNA and ABH2–dsDNA complexes, stabilized by a chemical cross-linking strategy. This study reveals that AlkB uses an unprecedented base-flipping mechanism to access the damaged base: it squeezes together the two bases flanking the flipped-out one to maintain the base stack, explaining the preference of AlkB for repairing ssDNA lesions over dsDNA ones. In addition, the first crystal structure of ABH2, presented here, provides a structural basis for designing inhibitors of this human DNA repair protein.
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Protein Data Bank
Atomic coordinates are deposited in Protein Data Bank under accession numbers 3BKZ ((Mn/2KG) AlkB–DNA1), 3BI3 ((Mn/2KG) AlkB–DNA5), 3BIE ((Mn/2KG)AlkB–DNA4), 3BTX (ABH2–DNA2), 3BTZ (ABH2–DNA3), 3BU0 ((Mn/2KG)ABH2–DNA2), 3BTY (ABH2–DNA6) and 3BUC ((Mn/2KG)ABH2–DNA6).
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We thank: R. Zhang and other beamline staff for assistance with data collection; Y. Luo and the Proteomics and Informatics Services Facility at Research Resources Center (University of Illinois at Chicago) for liquid chromatography–mass spectrometry analysis; and X. Yang, H. Chen and P. R. Chen for discussions. We also thank T. Lindahl and B. Sedgwick for the gift of the abh2 gene. This work was supported by National Institutes of Health (GM071440 to C.H. and a PCBio fellowship for C.T.S.), the W. M. Keck Foundation Distinguished Young Scholar in Medical Research Program (C.H.), and the Arnold and Mabel Beckman Foundation Young Investigator Program (C.H.). Data collection was performed at beamlines 19BM (Structure Biology Center) and 14BM (BioCARS) at the Advanced Photon Source at Argonne National Laboratory; financial support for these beamlines comes from the National Institutes of Health and the United States Department of Energy.
Author Contributions C.-G.Y. and C.Y. solved all AlkB–dsDNA and ABH2–dsDNA structures with help from E.M.D. (crystallography), C.T.S. (initial construct of ABH2 and crystallography) and X.J. (biochemistry). P.A.R. contributed to protein crystallography. C.H. designed the overall project and wrote the manuscript with C.-G.Y. and C.Y. All authors discussed results and commented on the manuscript.
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