ENDONUCLEASE V EXPRESSION IN HUMAN CELLS

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The eukaryotic DNA repair pathway involved in the repair of UV-induced lesions is thought to be at least as complex as the E. coli UvrA,B,C system. This complexity is inferred in the identification of ten different xeroderma pigmentosum (XP) complementation groups deficient in incision at UV-induced lesions. In contrast, endonuclease V encoded by the denV gene of bacteriophage T4, a pyrimidine dimer-DNA glycosylase with associated apyrimidinic endonuclease activity, has the capacity to initiate DNA repair as a single enzyme by incising specifically at pyrimidine dimers. The denV gene has been identified, sequenced and cloned in Escherichia coli, facilitating construction of denV plasmid vectors suitable for introduction into eukaryotic cells, including XP cells. XP cells have been instrumental in elucidating the effects of UV-induced photoproducts on biological processes due to their inability to repair them. During our study of denV- encoded endonuclease V-catalyzed UV repair in eukaryotic cells, three novel and interesting experimental observations have been made: (i) although UV excision repair and reporter gene expression can be restored to near normal levels by denV-encoded endonuclease V, cell survival is not restored to wild type; (ii) in contrast to the endogenous repair, which can be nonrandom and restricted to active genes, endonuclease V-catalyzed repair occurs throughout the entire genome; and (iii) the nucleotide patch size removed during endonuclease V-catalyzed pyrimidine dimer repair is much smaller than that in UV-irradiated normal cells. Primarily, two photoproducts, the abundant pyrimidine dimer and less abundant (6-4)TC pyrimidine-pyrimidone photoproducts [6-4]photoproducts), have been implicated in the diverse biological effects of UV light. Because neither pyrimidine dimers nor (6- 4)photoproducts are removed by XP cells, it has not been possible to unequivocally distinguish their individual contributions to the biological consequences of UV damage in vivo. Recently an XP revertant cell line has been described which repairs (6-4)TC photoproducts but not the pyrimidine dimers. Introducing the denV gene into XP and revertant XP cells provides an opportunity to separately analyze the effects of pyrimidine dimers and (6-4)TC photoproducts on DNA synthesis, transcription, survival, repair and mutations in human cells; and affords as the opportunity to examine the function of a well characterized DNA repair enzyme in normal and repair- deficient human cells.