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Suicide inactivation of the uracil DNA glycosylase UdgX by covalent complex formation

Nature Chemical Biology volume 15, pages 615–622 (2019)Cite this article

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Abstract

A uracil DNA glycosylase (UDG) from Mycobacterium smegmatis (MsmUdgX) shares sequence similarity with family 4 UDGs and forms exceedingly stable complexes with single-stranded uracil-containing DNAs (ssDNA-Us) that are resistant to denaturants. However, MsmUdgX has been reported to be inactive in excising uracil from ssDNA-Us and the underlying structural basis is unclear. Here, we report high-resolution crystal structures of MsmUdgX in the free, uracil- and DNA-bound forms, respectively. The structural information, supported by mutational and biochemical analyses, indicates that the conserved residue His109 located on a characteristic loop forms an irreversible covalent linkage with the deoxyribose at the apyrimidinic site of ssDNA-U, thus rendering the enzyme unable to regenerate. By proposing the catalytic pathway and molecular mechanism for MsmUdgX, our studies provide an insight into family 4 UDGs and UDGs in general.

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Fig. 1: The overall structure of MsmUdgX in the apo form.
Fig. 2: The overall structures of MsmUdgX in the uracil-bound form.
Fig. 3: Interactions of MsmUdgX with the DNA substrate in the complex.
Fig. 4: The cocrystal structures of the H109A and H109Q.
Fig. 5: The intramolecular interactions between motifs 1 and 3.

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Data availability

The atomic coordinates were deposited in the PDB with the accession numbers 6IO9, 6IOA, 6IOB, 6IOC and 6IOD. The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank J. Cox (UCSF) for providing the M. smegmatis strain, the BL19U1 beamline at Shanghai Synchrotron Radiation Facility for assistance during diffraction data collection, and Y. Ju, H. Zhou and Q. Jia for assistance with data collection and analyses in TSAs.

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Authors and Affiliations

  1. MOE Key Laboratory of Gene Function and Regulation, State Key Laboratory for Biocontrol, School of Life Sciences, The Sun Yat-Sen University, Guangzhou, Guangdong, China

    Jie Tu, Ran Chen & Wei Xie

  2. Department of Genetics and Biochemistry, Clemson University, Clemson, SC, USA

    Ye Yang & Weiguo Cao

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Contributions

W.X. conceived and designed the research. J.T., R.C. and Y.Y. performed the research. W.X. analyzed the data and wrote the paper. W.C. provided input into the data analysis and manuscript preparation. All authors reviewed the manuscript. Funding for this work was provided by the National Natural Science Foundation of China (grant nos. 31870782 and 31700657), the Foundation of State Key Laboratory for Biocontrol (grant no. SKLBC16KF02), the Natural Science Foundation of Guangdong province (grant no. 2018A030313313) and the NIH (grant no. GM121997).

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Correspondence to Wei Xie.

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Tu, J., Chen, R., Yang, Y. et al. Suicide inactivation of the uracil DNA glycosylase UdgX by covalent complex formation. Nat Chem Biol 15, 615–622 (2019). https://doi.org/10.1038/s41589-019-0290-x

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