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

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 N7guanine 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.

Astragalus polysaccharide protects formaldehyde-induced toxicity by promoting NER pathway in bone marrow mesenchymal stem cells

“…It has even been reported that DPCs is a molecular marker of FA-related cancer risk [11,25]. DPCs are necessary for DNA replication, transcription, and cell growth in normal cells; however, excessive DPCs produced by endogenous and exogenous agents block DNA replication cause genomic instability and tumorigenesis [26][27][28]. In this study, the degree of genotoxicity in BMSCs exposed to FA was studied by detecting the levels of DNA breakage, DPCs, and micronucleus formation.…”

Enzymatic bypass of an N6-deoxyadenosine DNA–ethylene dibromide–peptide cross-link by translesion DNA polymerases

“…TLS polymerases have been shown to bypass a GSHbased adduct (52,69,70) and (N-terminal-linked) peptides as long as 5-and 12-mers (71-73) but not a 23-mer peptide or green fluorescent protein (72). In vitro studies, including some in cells, have shown the mutagenic bypass of DNA-peptide cross-link by TLS polymerases (11,42,74). It is important to know how a DNA polymerase can bypass each cross-linked peptide entity and potentially cause base-pair mutations.…”

DNA-Histone Cross-Links: Formation and Repair

“…These super-bulky adducts can be generated by exposure of cells to endogenous and exogenous cross-linking agents (Stingele et al, 2017;Zhang et al, 2020). The presence of protein covalently attached to DNA strongly interferes with DNA replication, transcription, repair, and chromatin remodeling (Kuo et al, 2007;Klages-Mundt and Li, 2017;Yudkina et al, 2018;Ji et al, 2019). DPCs may be classified into five types, according to the nature of the covalent link in the DNA-protein complex and the presence of DNA strand breaks (Ide et al, 2015(Ide et al, , 2018Nakano et al, 2017).…”