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An unprecedented nucleic acid capture mechanism for excision of DNA damage

Abstract

DNA glycosylases that remove alkylated and deaminated purine nucleobases are essential DNA repair enzymes that protect the genome, and at the same time confound cancer alkylation therapy, by excising cytotoxic N3-methyladenine bases formed by DNA-targeting anticancer compounds. The basis for glycosylase specificity towards N3- and N7-alkylpurines is believed to result from intrinsic instability of the modified bases and not from direct enzyme functional group chemistry. Here we present crystal structures of the recently discovered Bacillus cereus AlkD glycosylase in complex with DNAs containing alkylated, mismatched and abasic nucleotides. Unlike other glycosylases, AlkD captures the extrahelical lesion in a solvent-exposed orientation, providing an illustration for how hydrolysis of N3- and N7-alkylated bases may be facilitated by increased lifetime out of the DNA helix. The structures and supporting biochemical analysis of base flipping and catalysis reveal how the HEAT repeats of AlkD distort the DNA backbone to detect non-Watson–Crick base pairs without duplex intercalation.

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Figure 1: Base excision repair of alkylated DNA by AlkD.
Figure 2: Crystal structures of AlkD in complex with 3d3mA-DNA and THF-DNA
Figure 3: Recognition of DNA damage by AlkD.
Figure 4: Excision of N 7- and O 2 -pyridyloxobutyl (POB) base adducts by AlkD.
Figure 5: Remodelling of a G•T wobble base pair by AlkD.

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Protein Data Bank

Data deposits

Atomic coordinates and structure factors for the reported crystal structures have been deposited with the Protein Data Bank under accession codes 3JX7 (3d3mA·T), 3JXY (G·T), 3JXZ (THF·T) and 3JY1 (THF·C).

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Acknowledgements

We thank J. Stivers for providing the pyrene phosphoramidite, Z. Warzak and LS-CAT beamline staff at the Advanced Photon Source (APS) for assistance with X-ray data collection, and T. Ellenberger, J. Stivers and P. O’Brien for comments on the manuscript. Use of the APS was supported by the US Department of Energy Office of Basic Energy Sciences. Use of LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor. This research was supported by a grant from the American Cancer Society (to B.F.E.) and the NIH (RO1 CA29088 to B.G.). E.H.R. was supported in part by the Vanderbilt Training Program in Molecular Toxicology. Additional support for local crystallography facilities was provided by the Vanderbilt Center in Molecular Toxicology and the Vanderbilt-Ingram Cancer Center.

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Contributions

E.H.R. purified and crystallized AlkD, determined crystal structures and performed 7mG activity assays; B.G. synthesized 3d3mA oligonucleotides; A.S.P.G. and T.E.S. performed POB activity assays; B.F.E. designed the project; B.F.E. and E.H.R. analysed data and wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Brandt F. Eichman.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Tables 1-2, Supplementary Text comprising information on 3-deaza-3-methyladenine as a 3mA mimetic, AlkD does not discriminate against the base opposite the lesion, AlkD traps and restructures destabilized base pairs, Base excision by solvent exposure and a Discussion onExtrusion of DNA bases without duplex intercalation. The file also contains additional references and figures 1-12 with legends. (PDF 6429 kb)

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Rubinson, E., Gowda, A., Spratt, T. et al. An unprecedented nucleic acid capture mechanism for excision of DNA damage. Nature 468, 406–411 (2010). https://doi.org/10.1038/nature09428

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