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Lack of DNA enzymatic photoreactivation in HeLa cell‐free extracts
Author(s) -
Chao Chuck C.-K.
Publication year - 1993
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(93)80848-o
Subject(s) - photolyase , hela , biology , dna , dna repair , microbiology and biotechnology , saccharomyces cerevisiae , nucleic acid , biochemistry , yeast , chloramphenicol acetyltransferase , gene , cell , gene expression , reporter gene
Using a DNA‐protein binding assay, we have previously identified and characterized a UV‐damaged DNA recognition protein (UVDRP) from HeLa cells [(1991) Nucleic Acids Res. 19, 6413‐6418]. In this report, the photoreactivating activity of UVDRP from the yeast, Saccharomyces cerevisiae, and HeLa cells was investigated. Although yeast and human cells are evolutionarily different from each other, both UVDRPs were conserved in the sense of their biochemical characteristics except that the yeast UVDRP also exhibited an enzymatic photoreactivating activity. A mammalian expression vector plasmid DNA carrying the bacterial chloramphenicol acetyltransferase (CAT) gene was UV irradiated in vitro followed immediately by exposure to photoreactivating light, and its transient expression in repair‐deficient xeroderma pigmentosum (XP) cells was investigated. More than 80% of the CAT activity was inhibited by UV irradiation, which was partially restored (> 60%) by a partially purified yeast photolyase. In contrast, HeLa cell extracts did not express a photoreactivatable recovery from UV‐induced inhibition of the CAT activity tested in the same system. This study has demonstrated the potential use of the DNA‐mobility shift assay to investigate enzymatic photoreactivation, and has indicated the absence of the repair mechanism in human cells.