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Covalent binding of uracil DNA glycosylase UdgX to abasic DNA upon uracil excision

Abstract

Uracil DNA glycosylases (UDGs) are important DNA repair enzymes that excise uracil from DNA, yielding an abasic site. Recently, UdgX, an unconventional UDG with extremely tight binding to DNA containing uracil, was discovered. The structure of UdgX from Mycobacterium smegmatis in complex with DNA shows an overall similarity to that of family 4 UDGs except for a protruding loop at the entrance of the uracil-binding pocket. Surprisingly, H109 in the loop was found to make a covalent bond to the abasic site to form a stable intermediate, while the excised uracil remained in the pocket of the active site. H109 functions as a nucleophile to attack the oxocarbenium ion, substituting for the catalytic water molecule found in other UDGs. To our knowledge, this change from a catalytic water attack to a direct nucleophilic attack by the histidine residue is unprecedented. UdgX utilizes a unique mechanism of protecting cytotoxic abasic sites from exposure to the cellular environment.

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Fig. 1: Overall structure of MsmUdgX.
Fig. 2: MsmUdgX–5-mer ssDNA complex.
Fig. 3: Covalent bond formation by H109 of the signature motif.
Fig. 4: Snapshot of the stable covalent intermediate.
Fig. 5: Assay of uracil excision and binding by MsmUdgX and mutant proteins.

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Data availability

The crystallographic data that support the findings of this study are available from the PDB (https://www.rcsb.org/) under the following accession codes: Apo-UdgX, 6AIL; UdgX–5-mer ssDNA complex, 6AJO; UdgX–dUMP complex, 6AJP; UdgX–uracil complex, 6AJR; UdgX H109S mutant, 6AJS; and UdgX E52Q mutant, 6AJQ.

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Acknowledgements

This research was partly supported by the Marine Biotechnology Program of the Korea Institute of Marine Science and Technology Promotion (KIMST), by the Ministry of Oceans and Fisheries (MOF) (no. 20170488) and partially by the National Research Fund (NRF-2018R1A2A2A05021648) and KRIBB Research Initiative. To B.-H.O., the work was supported by the National Research Fund (NRF-2018R1A2B3004764). In the laboratory of U.V., the work was supported by grants from the Department of Science and Technology (DST) and the Department of Biotechnology (DBT), New Delhi, India. U.V. also acknowledges the DBT-IISc partnership program, University Grants Commission, New Delhi, for the Centre of Advanced Studies and DST-FIST level II infrastructure support for this work. U.V. is a J. C. Bose fellow of the DST, New Delhi, India.

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W.-C.A. and M.-H.L. performed crystallography; W.-C.A., S.A. and P.B.S. performed biochemical assays; J.H.M. and G.S.L. performed MS; W.-C.A., S.A., J.-H.K., J.H.M., B.-H.O., U.V. and E.-J.W. designed the research and analyzed data; W.-C.A., U.V. and E-J.W. wrote the manuscript.

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Correspondence to Umesh Varshney or Eui-Jeon Woo.

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Ahn, WC., Aroli, S., Kim, JH. et al. Covalent binding of uracil DNA glycosylase UdgX to abasic DNA upon uracil excision. Nat Chem Biol 15, 607–614 (2019). https://doi.org/10.1038/s41589-019-0289-3

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