DNA-protein cross-links (DPCs) are unusually bulky DNA adducts that block the access of proteins to DNA and interfere with gene expression, replication, and repair. We previously described DPC formation at the N7-guanine position of DNA in human cells treated with antitumor nitrogen mustards and platinum compounds and have shown that DPCs can form endogenously at DNA epigenetic mark 5-formyl-dC. However, insufficient information is available about the effects of these structurally distinct DPCs on transcription. In the present work, we employ a combination of in vitro assays, mass spectrometry, and molecular dynamics simulations to examine the ability of phage T7 RNA polymerase to bypass DPCs conjugated to the C7 position of 7-deaza-dG and the C5 position of dC. These model adducts represent endogenous DPCs induced by exposure to antitumor drugs and formed at epigenetics DNA marks, respectively. Our results reveal that DPCs containing full-length proteins significantly inhibit in vitro transcription by T7 RNA polymerase, while short DNA-peptide cross-links (DpCs) are bypassed. DpCs conjugated to the C7 position of 7-deaza-dG are transcribed with high fidelity, while the same polypeptides attached to the C5 position of dC induce transcription errors. Molecular dynamics simulations of DpCs conjugated either to the C5 atom of dC or the C7 position of 7-deaza-dG on the template strand in T7 RNA polymerase explain how the conjugated peptide can be accommodated in the narrow major groove of the DNA-RNA hybrid and how the modified dC can form a stable mismatch with the incoming ATP in the polymerase active site, allowing for transcriptional mutagenesis.
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.9b00365 . Methods (MD parameters for nonstandard residues, structural stability, structural clustering, major groove width); supplementary tables; and supplementary figures ( PDF ) Movie S1: T7 RNA polymerase ternary complex, containing the 11mer peptide cross-linked to the templating dC opposite to the N1 protonated incoming ATP ( AVI ) National Institute of Environmental Health Sciences [R01 ES-023350 to N.T., R01 ES-025987 to S.B.]; National Cancer Institute [R01 CA-075449 to S.B.]; Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by the National Science Foundation [MCB060037 to S.B.]; Wayland E. Noland Graduate Student Fellowship and Doctoral Dissertation Fellowship at the University of Minnesota to S.J. (in part). The authors declare no competing financial interest.
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