Abstract
Using a positive selection system for isolating DNA replication and repair related mutants, we isolated a clone from a rat kangaroo cell line (PtK2) that has increased sensitivity to UV light. Characterization of this clone indicated normal post-replication repair after UV irradiation, and normal removal rates of cyclobutane pyrimidine dimers and pyrimidine(6-4)pyrimidone photoproducts by excision repair. However, this cell line has decreased ability to make early incisions on damaged DNA, possibly indicating a defect in preferential repair of actively transcribed genes, and a slower cell proliferation rate, including a longer S-phase. This phenotype reinforces the present notion that control of key mechanisms in cell metabolism, such as cell cycle control, repair, transcription and cell death, can be linked.
Highlights
Isolation of DNA repair mutants from normal culture cells has proven to be very useful in identifying genes related to cell genome stability maintenance (Collins, 1993)
A positive selection procedure for isolating cell mutants potentially affected in their ability to process DNA damage was tested
11 clones were no different from parental cell line; three, more resistant to UV, and the remainder slightly more UV sensitive
Summary
Isolation of DNA repair mutants from normal culture cells has proven to be very useful in identifying genes related to cell genome stability maintenance (Collins, 1993). A photoreactivation prone cell line (PtK2) from the marsupial Potorous tridactylus (Chiang and Rupert, 1979), is useful for isolating mutants defective in processing ultraviolet (UV)-induced DNA damage, with a positive selection system similar to that described by Rosenstein and Ohlsson-Wilhelm (1979) in ICR 2A frog cells. This system uses cell ability to eliminate cyclobutane pyrimidine dimers (CPD) enzymatically through exposure to light (300-500 nm) by photoreactivation, to rescue mutant cells unable to process DNA damages by incorporating the photosensitizer nucleotide analogue 5-bromo-2’-deoxyuridine (BUdr). A detailed characterization of this clone concerning nucleotide excision and post-replication repair was made and is described below
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