Duplex interrogation by a direct DNA repair protein in search of base damage

Abstract

ALKBH2 is a direct DNA repair dioxygenase guarding the mammalian genome against N1-methyladenine, N3-methylcytosine and 1,N6-ethenoadenine damage. A prerequisite for repair is to identify these lesions in the genome. Here we present crystal structures of human ALKBH2 bound to different duplex DNAs. Together with computational and biochemical analyses, our results suggest that DNA interrogation by ALKBH2 has two previously unknown features: (i) ALKBH2 probes base-pair stability and detects base pairs with reduced stability, and (ii) ALKBH2 does not have nor need a damage-checking site, which is critical for preventing spurious base cleavage for several glycosylases. The demethylation mechanism of ALKBH2 insures that only cognate lesions are oxidized and reversed to normal bases, and that a flipped, non-substrate base remains intact in the active site. Overall, the combination of duplex interrogation and oxidation chemistry allows ALKBH2 to detect and process diverse lesions efficiently and correctly.

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Figure 1: Base pairs with different stability are discernible by ALKBH2.
Figure 2: Contributions of ALKBH2 to base flipping in duplex DNA.
Figure 3: ALKBH2 probes the stability of a base pair to detect DNA damage.
Figure 4: ALKBH2 does not have nor need a damage-checking site; its oxidation chemistry insures that non-substrate bases are not modified.

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Acknowledgements

This study was supported by the US National Institutes of Health (GM071440 to C.H.), the Hundred Talent Program of the Chinese Academy of Sciences (to C.-G.Y.), the State Key Development Program of Basic Research of China (2009CB918502 to C.-G.Y.), the special grant for Stem Cell and Regenerative Medicine (XDA01040305 to C.-G.Y.), the National Science Foundation (MCB-0547854 to A.R.D.), Beamlines 23ID-B (General Medicine and Cancer Institutes Collaborative Access Team, GM/CA-CAT), 21ID-D (Life Sciences Collaborative Access Team, LS-CAT) and 24ID-E (The Northeastern Collaborative Access Team, NE-CAT) at the Advanced Photon Source at Argonne National Laboratory, the Shanghai Synchrotron Radiation Facility (BL17U1) and the US Department of Energy.

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C.Y., C.-G.Y. and C.H. designed the experiments. Experiments were conducted by C.Y., B.C., W.Z., G.J., L.Z. and C.J.L.; computational analyses were carried out by B.Q. and A.R.D. C.Y. and C.H. wrote the paper, and B.Q., A.R.D. and C.-G.Y. contributed to editing the manuscript.

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Correspondence to Cai-Guang Yang or Chuan He.

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Yi, C., Chen, B., Qi, B. et al. Duplex interrogation by a direct DNA repair protein in search of base damage. Nat Struct Mol Biol 19, 671–676 (2012). https://doi.org/10.1038/nsmb.2320

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