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Adriamycin-mediated introduction of a limited number of single-strand breaks into supercoiled DNA
Abstract:
It was reported previously that Adriamycin converts form I covalently closed circular, supercoiled bacteriophage PM2 DNA to the relaxed circular form II DNA; no form III linear DNA was produced as a result of the extracellular action of Adriamycin in the presence of NADH-dehydrogenase. When form II DNA, produced by the action of Adriamycin, was treated with the BAL 31 nuclease, a single sharp DNA band after agarose gel electrophoresis indicated the presence of only full-length linear form III DNA. As one of its activities, the BAL 31 nuclease introduces a single-strand break in the complementary strand opposite a preexisting single-strand break. When form II DNA, produced by the action of gamma irradiation, was reacted with the BAL enzyme, the resulting linear DNA molecules exhibited a broad range of molecular weights, indicating the presence of many single-strand breaks in the substrate form II DNA. When the Adriamycin-produced form II DNA was treated with restriction endonucleases that cleave PM2 DNA at a single site, either with or without pretreatment with the BAL enzyme, the formation of only full-length linear DNA was observed. Thus, the drug is capable of introducing one or only a very limited number of single-strand breaks into supercoiled DNA; furthermore, these breaks are introduced at random sites along the DNA molecules.
Insights
Adriamycin introduces a limited number of random single-strand breaks into supercoiled DNA, converting it to relaxed circular DNA. Further treatment with BAL 31 nuclease or restriction enzymes confirms these breaks are site-specific and few.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Adriamycin is known to interact with DNA.
- Supercoiled DNA (form I) can be converted to relaxed circular DNA (form II).
- BAL 31 nuclease and restriction enzymes are tools for DNA analysis.
Purpose of the Study:
- To investigate the mechanism by which Adriamycin affects supercoiled bacteriophage PM2 DNA.
- To characterize the nature and number of DNA breaks induced by Adriamycin.
- To compare Adriamycin-induced DNA modification with other DNA-damaging agents.
Main Methods:
- Agarose gel electrophoresis to analyze DNA forms (I, II, III).
- Treatment of Adriamycin-modified DNA with BAL 31 nuclease.
- Treatment of Adriamycin-modified DNA with restriction endonucleases.
- Comparison with DNA modified by gamma irradiation.
Main Results:
- Adriamycin converts form I to form II DNA without producing linear form III DNA.
- BAL 31 nuclease treatment of Adriamycin-induced form II DNA yields only full-length linear form III DNA.
- Restriction endonuclease digestion of Adriamycin-induced form II DNA also yields only full-length linear DNA, indicating limited breaks.
- Gamma-irradiated form II DNA shows a broad range of linear DNA fragments, indicating multiple breaks.
Conclusions:
- Adriamycin induces one or a very limited number of single-strand breaks in supercoiled DNA.
- These breaks are introduced at random sites along the DNA molecule.
- The action of Adriamycin on DNA differs significantly from agents like gamma irradiation that cause multiple, random breaks.