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.
Highlights
A wide variety of DNA lesions such as ultraviolet light-induced photoproducts and chemically induced bulky adducts and crosslinks interfere with replication and lead to mutations and cell death
We found that only this combination of in vitro transcription and real-time reverse transcription polymerase chain reaction (PCR) can detect both cisplatin- and ultraviolet light (UV)-induced DNA lesions that interfere with transcription
Inhibition of transcription can be effected by RNA polymerase stalling due to DNA lesions or RNA polymerase II inhibitors such as alpha-amanitin from the death cap [8, 9] and actinomycin D from Streptomyces bacteria, which intercalate into DNA and activate an inducer of apoptosis in many cell lines [10, 11]
Summary
A 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. 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. The induced DNA lesions interfere with replication, leading to mutations and cell death, and have been associated with cancer, inborn diseases, and aging. DNA lesions interfere with transcription, inhibiting elongation by RNA polymerase and leading to reduced transcription and/or mutations of the transcript [3, 4]. Inhibition of transcription can be effected by RNA polymerase stalling due to DNA lesions or RNA polymerase II inhibitors such as alpha-amanitin from the death cap [8, 9] and actinomycin D from Streptomyces bacteria, which intercalate into DNA and activate an inducer of apoptosis in many cell lines [10, 11].
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