An in vitro method for detecting genetic toxicity based on inhibition of RNA synthesis by DNA lesions

Abstract

IntroductionA wide variety of DNA lesions such as ultraviolet light-induced photoproducts and chemically induced bulky adducts and crosslinks (intrastrand and interstrand) interfere with replication and lead to mutations and cell death. In the human body, these damages may cause cancer, inborn diseases, and aging. So far, mutation-related actions of DNA polymerases during replication have been intensively studied. However, DNA lesions also block RNA synthesis, making the detection of their effects on transcription equally important for chemical safety assessment. Previously, we established an in vivo method for detecting DNA damage induced by ultraviolet light and/or chemicals via inhibition of RNA polymerase by visualizing transcription.ResultsHere, we present an in vitro method for detecting the effects of chemically induced DNA lesions using in vitro transcription with T7 RNA polymerase and real-time reverse transcription polymerase chain reaction (PCR) based on inhibition of in vitro RNA synthesis. Conventional PCR and real-time reverse transcription PCR without in vitro transcription can detect DNA lesions such as complicated cisplatin DNA adducts but not UV-induced lesions. We found that only this combination of in vitro transcription and real-time reverse transcription PCR can detect both cisplatin- and UV-induced DNA lesions that interfere with transcription.ConclusionsWe anticipate that this method will be useful for estimating the potential transcriptional toxicity of chemicals in terminally differentiated cells engaged in active transcription and translation but not in replication.

Interaction Analysis of T7 RNA Polymerase with Heparin and Its Low Molecular Weight Derivatives – An in Silico Approach

“…Such RNAs could also be useful as hybridization probes, mRNAs for in vitro translation, substrates for analyzing the processing reactions or RNA splicing, or for any purpose requiring a specific RNA. 2 Apart from the use in selective high-level expression of cloned genes, T7 RNAP serves as a template for multiple purposes such as detecting the effects of chemically induced DNA lesions, 3 creating synthetic gene circuits, 4 and RNA editing. 5 T7RNAP is a 99 kDa single polypeptide DNA-dependent RNA polymerase, which catalyzes the formation of RNA in 5′-3′ direction.…”

Experimental evolution of UV resistance in a phage

“…It is well established that cis–syn cyclobutane pyrimidine dimers, pyrimidine:pyrimidone 6,4, photoproducts, and other UV-induced DNA damage block transcription by E. coli and T7 RNAP when the lesions are present on the transcribed strand ( Sonohara, Iwai & Kuraoka, 2015 ). However, the lesions have little to no effect when present on the non-template strand.…”

Effects of acetaldehyde-induced DNA lesions on DNA metabolism

“…For in vitro transcription assay [15], 50 μL reactions containing 100 ng DNA template, 4 mM NTP mixture (ATP, CTP, GTP, and UTP) and 5 units thermo T7 RNA polymerase in buffer (40 mM Tris-HCl, pH 8.0, 50 mM NaCl, 8 mM MgCl2, 5 mM dithiothreitol and 20 units RNase inhibitor) were incubated at 37°C for 1 h. RNA transcripts were purified using an RNeasy Mini Kit (QIAGEN) with RNase-Free DNase (QIAGEN) according to the manufacturer's instructions. Next, cDNAs were generated from purified RNA samples using primer 2440-2421 (5′-gcggccaacttacttctgac-3′) and ReverTra Ace reverse transcriptase (TOYOBO) according to the manufacturer's instructions.…”

“…Previously, we reported an in vitro method for detecting effects of chemically induced DNA lesions using in vitro transcription with T7 RNA polymerase and real-time reverse transcription polymerase chain reaction (PCR), based on inhibition of in vitro RNA synthesis (Fig. 4a) [15]. This assay was used for acetaldehyde.…”