Transcriptional Bypass of DNA-Protein and DNA-Peptide Conjugates by T7 RNA Polymerase

Shaofei Ji, Jenna Thomforde, Colette Rogers, Iwen Fu, Suse Broyde, Natalia Y. Tretyakova

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
JournalACS Chemical Biology
DOIs
StateAccepted/In press - Jan 1 2019

Fingerprint

Peptides
DNA
Proteins
Transcription
bacteriophage T7 RNA polymerase
Molecular Dynamics Simulation
Epigenomics
Nitrogen Mustard Compounds
Molecular dynamics
Platinum Compounds
Bacteriophage T7
Mutagenesis
Bacteriophages
DNA Adducts
Computer simulation
Guanine
link protein
Gene expression
Antineoplastic Agents
Mass spectrometry

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Medicine

Cite this

Transcriptional Bypass of DNA-Protein and DNA-Peptide Conjugates by T7 RNA Polymerase. / Ji, Shaofei; Thomforde, Jenna; Rogers, Colette; Fu, Iwen; Broyde, Suse; Tretyakova, Natalia Y.

In: ACS Chemical Biology, 01.01.2019.

Research output: Contribution to journalArticle

Ji, Shaofei ; Thomforde, Jenna ; Rogers, Colette ; Fu, Iwen ; Broyde, Suse ; Tretyakova, Natalia Y. / Transcriptional Bypass of DNA-Protein and DNA-Peptide Conjugates by T7 RNA Polymerase. In: ACS Chemical Biology. 2019.
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