Lack of DNA enzymatic photoreactivation in HeLa cell-free extracts.

Chuck C.-K. Chao

Research output: Contribution to journalJournal Article peer-review

Abstract

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.
Original languageAmerican English
Pages (from-to)411-416
JournalFEBS Letters
Volume336
Issue number3
DOIs
StatePublished - 1993

Keywords

  • Cell Line
  • Cell Nucleus/metabolism
  • Cell-Free System
  • Chromatography, Affinity
  • DNA Damage
  • DNA Repair
  • DNA, Fungal/metabolism
  • DNA, Neoplasm/metabolism
  • DNA-Binding Proteins/isolation & purification
  • DNA-Binding Proteins/metabolism
  • Deoxyribodipyrimidine Photo-Lyase/metabolism
  • Fungal Proteins/isolation & purification
  • Fungal Proteins/metabolism
  • HeLa Cells
  • Humans
  • Saccharomyces cerevisiae/radiation effects
  • Ultraviolet Rays
  • Xeroderma Pigmentosum

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