The enzyme uracil DNA glycosylase (UNG) excises unwanted uracil bases in the genome using an extrahelical base recognition mechanism. Efficient removal of uracil is essential for prevention of C-to-T transition mutations arising from cytosine deamination, cytotoxic U•A pairs arising from incorporation of dUTP in DNA, and for increasing immunoglobulin gene diversity during the acquired immune response. A central event in all of these UNG-mediated processes is the singling out of rare U•A or U•G base pairs in a background of approximately 109 T•A or C•G base pairs in the human genome. Here we establish for the human and Escherichia coli enzymes that discrimination of thymine and uracil is initiated by thermally induced opening of T•A and U•A base pairs and not by active participation of the enzyme. Thus, base-pair dynamics has a critical role in the genome-wide search for uracil, and may be involved in initial damage recognition by other DNA repair glycosylases.
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We thank A. Majumdar for assistance with NMR experiments. This work was supported by NIH grants (J.T.S. and L.M.A.) and a major research instrumentation grant from the NSF.
Author Contributions J.B.P. prepared all mutant enzymes and performed the NMR experiments; M.A.B. collected, processed and refined X-ray data and performed structural analyses; D.J.K. conceived the structural approach and purified and crystallized the complexes; J.I.F. performed NMR studies on the L272G mutant; L.M.A. performed structural analyses, discussed and commented on the manuscript; J.T.S. analysed data and wrote the paper.
Coordinates and structure factor files for the EI complex and the abasic DNA complex have been deposited in the Protein Data Bank with accession numbers 2OXM and 2OYT, respectively.
Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.
This file contains Supplementary Figures 1-4 with Legends and Supplementary Tables 1-3. The Supplementary Figures contain: (1) a scheme describing the general findings, (2) electron density maps of the flipped thymine and the abasic sugar excision product, (3) imino proton spectra of the free T/A 10 mer DNA duplex and its complexes with wild-type and mutant UNG enzymes, and (4) magnetization transfer time courses for the imino protons of residues G3, G4 and T2 of free and UNG-bound DNA. The Supplementary Tables detail (1) crystallographic data collection and refinement statistics, (2) imino proton exchange rates of free and bound DNA, and (3) chemical shifts of imino protons in each enzyme complex. (PDF 1951 kb)
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Parker, J., Bianchet, M., Krosky, D. et al. Enzymatic capture of an extrahelical thymine in the search for uracil in DNA. Nature 449, 433–437 (2007). https://doi.org/10.1038/nature06131
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