Mechanism of transcriptional stalling at cisplatin-damaged DNA

Abstract

The anticancer drug cisplatin forms 1,2-d(GpG) DNA intrastrand cross-links (cisplatin lesions) that stall RNA polymerase II (Pol II) and trigger transcription-coupled DNA repair. Here we present a structure-function analysis of Pol II stalling at a cisplatin lesion in the DNA template. Pol II stalling results from a translocation barrier that prevents delivery of the lesion to the active site. AMP misincorporation occurs at the barrier and also at an abasic site, suggesting that it arises from nontemplated synthesis according to an 'A-rule' known for DNA polymerases. Pol II can bypass a cisplatin lesion that is artificially placed beyond the translocation barrier, even in the presence of a G·A mismatch. Thus, the barrier prevents transcriptional mutagenesis. The stalling mechanism differs from that of Pol II stalling at a photolesion, which involves delivery of the lesion to the active site and lesion-templated misincorporation that blocks transcription.

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Figure 1: Structure of a cisplatin-damaged Pol II elongation complex.
Figure 2: Pol II stalling and misincorporation.
Figure 3: The cisplatin lesion is not stably accommodated in the active site.
Figure 4: Lesion bypass.
Figure 5: Different mechanisms of Pol II stalling at dinucleotide lesions.

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Acknowledgements

We thank members of the Cramer laboratory for help. P.C. and T.C. were supported by the Deutsche Forschungsgemeinschaft, the Sonderforschungsbereich SFB646 and the Fonds der chemischen Industrie. P.C. was supported by the EU grant 3D repertoire, contract no. LSHG-CT-2005-512028. G.E.D. and F.B. were supported by the Elite Netzwerk Bayern. A.A. was supported by the Marie Curie training and mobility network CLUSTOX DNA.

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G.E.D. performed and analyzed biochemical and crystallographic experiments. A.A. synthesized cisplatin-containing DNA strands and performed MALDI experiments. F.B. assisted with experiments and crystallography. T.C. supervised the project. P.C. supervised the project and wrote the manuscript.

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Correspondence to Thomas Carell or Patrick Cramer.

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Supplementary Figures 1–5, Supplementary Table 1 (PDF 1161 kb)

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Damsma, G., Alt, A., Brueckner, F. et al. Mechanism of transcriptional stalling at cisplatin-damaged DNA. Nat Struct Mol Biol 14, 1127–1133 (2007). https://doi.org/10.1038/nsmb1314

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