The object of this research is to understand the differential sensitivity of cells at different cell cycle stages following DNA damage. Since we have been unable to explain these effects in terms of different amounts of damage and/or repair, we plan to concentrate our efforts on indirect effects manifested through inhibition of DNA replication. Part of this project depends on the differential induction of different lesions at 254nm and 313nm light. By comparing the inhibition of DNA replication resulting from irradiation at these two wavelengths, we determined that lesions other than T4 endonuclease V sensitive sites caused perturbations in DNA synthesis. We hypothesize that the alkaline-labile pyrimidine-cytosine 6 yield 4 photoproduct described by Lippke et al (proc. Natl. Acad. Sci. 78:3388) may be responsible for this effect. Using a very sensitive radioimmunoassay developed by us, we intend to quantitate the induction and repair of these different lesions by changing the nature of the probe. Defined DNA sequences will be used to determine the specificity of the antisera and T4 endonuclease V, and reverse phase liquid chromatography will give additional clarification on induction of different dimers at each wavelength in cases where the antiserum shows cross-reactivity. The effects of these different lesions may be felt indirectly as a disruption in the order of replication of different DNA sequences. Using CHO MK 42 cells in which the dihydrofolate reductase gene is amplified, we intend to map the time of replication of this gene in normal and damaged cells. In this way we may be able to determine the importance of specific DNA lesions and get some insight into their mode of action.
| Mitchell, D L; Clarkson, J M; Chao, C C et al. (1986) Repair of cyclobutane dimers and (6-4) photoproducts in ICR 2A frog cells. Photochem Photobiol 43:595-7 |
| Mitchell, D L; Clarkson, J M; Adair, G M (1986) The DNA of UV-irradiated normal and excision-deficient mammalian cells undergoes relaxation in an initial stage of DNA repair. Mutat Res 165:123-8 |
| Mitchell, D L; Haipek, C A; Clarkson, J M (1985) (6-4)Photoproducts are removed from the DNA of UV-irradiated mammalian cells more efficiently than cyclobutane pyrimidine dimers. Mutat Res 143:109-12 |
| Mitchell, D L; Haipek, C A; Clarkson, J M (1985) Further characterisation of a polyclonal antiserum for DNA photoproducts: the use of different labelled antigens to control its specificity. Mutat Res 146:129-33 |
