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Transcription by T7 RNA polymerase using benzo[a]pyrene-modified templates
S T Nath1, L J Romano, L J Romana
1Department of Chemistry, Wayne State University, Detroit, MI 48202.
Carcinogenesis
|June 1, 1991
Summary
Bulky DNA adducts, like those from benzo[a]pyrene, significantly inhibit T7 RNA polymerase. Inhibition is much greater when adducts are on the DNA template strand, causing transcription to terminate.
Area of Science:
- Biochemistry
- Molecular Biology
- Toxicology
Background:
- DNA damage can impede cellular processes like transcription.
- Benzo[a]pyrene (B[a]P) is a potent carcinogen forming bulky DNA adducts.
- T7 RNA polymerase is a key enzyme for in vitro transcription.
Purpose of the Study:
- To investigate how bulky DNA adducts affect T7 RNA polymerase transcription.
- To compare the inhibitory effects of B[a]P adducts on different DNA strands.
Main Methods:
- Synthesized M13 DNA templates with single B[a]P adducts in either the template or displaced strand.
- Utilized in vitro transcription assays with T7 RNA polymerase.
- Analyzed transcription products using polyacrylamide gel electrophoresis.
Main Results:
- B[a]P adducts caused significant inhibition of T7 RNA polymerase activity.
- Adducts in the template strand resulted in at least 10-fold greater inhibition compared to the displaced strand.
- Template strand adducts led to discrete termination products, while displaced strand adducts yielded products similar to native DNA.
Conclusions:
- The position of bulky DNA adducts critically influences transcription inhibition by T7 RNA polymerase.
- Template strand adducts act as potent blocks to transcription, causing premature termination.
- Understanding these mechanisms is crucial for assessing the genotoxicity of carcinogens like B[a]P.