PMID: 2106726Jan 1, 1990Paper

On the DNA polymerase-a mutant: immunofluorescence assay of UV-induced thymidine dimers in Aphr-4-2 cells

Somatic Cell and Molecular Genetics
P K Liu, G S Hsu

Abstract

Aphidicolin inhibits purified DNA polymerases-a and -d in vitro and inhibits mitosis in animal cells. The Chinese hamster V79 cell mutant, Aphr-4-2, was selected for its ability to form colonies in cultured medium supplemented with 1.0 microM aphidicolin. At this concentration, the parental wild-type V79 cells (clone 743x) have a survival rate of less than 10(-7). The mutant DNA polymerase-a is resistant to aphidicolin at concentrations that are inhibitory to the wild-type V79 DNA polymerase-a. The apparent Km for dCTP of the mutant DNA polymerase-a is consistently lower than that of the wild-type DNA polymerase-a. This mutant exhibits slow growth, mutator activity, hypersensitivity, and hypermutability to UV. We wanted to know the basis of UV hypersensitivity in this mutant. Using the antisera (UV2) raised against UV-induced thymidine dimers and a sensitive immunofluorescence assay to measure UV-induced thymidine dimers and with detection in ACAS 570 Workstation, we observed that 50% of the thymidine dimers disappeared within 5 h after irradiation and more than 80% of the dimers were removed within 24 h in both cell lines. These results indicate that the recognition, incision, and excision steps in nucleotide excision repair p...Continue Reading

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Citations

Dec 17, 2009·Photochemistry and Photobiology·David Mitchell, Becky Brooks

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