ABSTRACT
The DNA-unwinding method developed by Ahnström and his coworkers to measure DNA strand breaks in mammalian cells was used to measure single-strand breaks (SSB) in the DNA of intact yeast cells. DNA unwinding, which took place inside the rigid cell wall of yeast, was investigated as a function of time, radiation dose, and of pH and salt concentration of the alkaline solution. After DNA unwinding had taken place, the cell wall was destroyed by partial enzymatic digestion and sonication in the presence of detergents. Fragments of single- and double-stranded DNA were separated using hydroxylapatite chromatography. In this way the most suitable conditions for DNA unwinding within the cell wall were established. The results show that SSB and double-strand breaks (DSB) give rise to different kinetics of DNA unwinding.
Subject(s)
DNA Repair/radiation effects , DNA, Fungal/radiation effects , DNA, Single-Stranded/radiation effects , Saccharomyces cerevisiae/radiation effects , DNA, Fungal/metabolism , DNA, Single-Stranded/metabolism , Kinetics , Methods , Nucleic Acid Denaturation , Saccharomyces cerevisiae/metabolismABSTRACT
The kinetics of disappearance of single-strand breaks (SSB) from the DNA of X-irradiated stationary yeast cells under liquid-holding conditions was found to proceed in a dose-independent manner up to a dose of at least 2400 Gy, and was found to be complete after incubation of cells for 1 h. This was deduced from data for a yeast wild-type (WT) haploid and diploid strain as well as for rad52 haploid cells defective in DNA double-strand break (DSB) repair. In all cases an initial fast repair component assumed to correspond to SSB repair was observed whereby about 80% of the induced 'unwinding points' disappeared from the DNA with a time constant of about 3 min. Following this fast component, a slower component of removal of 'unwinding points' occurred with a time constant estimated to be 20 min. The molecular nature of these two components of repair is not known. We could find no evidence for the induction of secondary (enzymatic) breaks in the DNA during post-irradiation incubation. Incubation of cells in growth medium after irradiation resulted in similar kinetics as those under liquid-holding conditions. In the absence of an energy source in the medium (i.e. when cells were incubated in buffer or distilled water after irradiation) only 60-80% of the SSB were removed from yeast DNA. Residual SSB disappeared from the DNA only when cells were transferred to a medium containing glucose. The relative mass of DNA unwound per induced strand break (i.e. represented by the slope of the dose-effect curve immediately after irradiation) was found to change slowly with the age of the cell culture under liquid-holding conditions. This effect had to be corrected for in the measurements of strand break repair under these conditions.
Subject(s)
DNA Repair/radiation effects , DNA, Fungal/radiation effects , DNA, Single-Stranded/radiation effects , Saccharomyces cerevisiae/radiation effects , Culture Media , DNA, Fungal/metabolism , DNA, Single-Stranded/metabolism , Kinetics , Methods , Nucleic Acid Denaturation , Saccharomyces cerevisiae/metabolism , TemperatureABSTRACT
Cyclophosphamide (CP), whether applied in its chemically activated form as 4-hydroperoxy-cyclophosphamide (4-OOH-CP) in vitro or in the host-mediated assay (HMA) using rats, exhibits toxic and mutagenic effects on excision deficient yeast cells. The expression of these effects is examined during a prolonged postincubation in buffer and compared with the ability of activated CP to induce interstrand cross-links and DNA fragmentation. At comparable doses, we observed a close similarity of biological and biochemical effects in either test system.
