title: Host cell reactivation of gene expression for an adenovirus-encoded reporter gene reflects the repair of UVC-induced cyclobutane pyrimidine dimers and methylene blue plus visible light-induced 8-oxoguanine.
abstract: Previously, we have reported the use of a recombinant adenovirus (Ad)-based host cell reactivation (HCR) assay to examine nucleotide excision repair (NER) of UVC-induced DNA lesions in several mammalian cell types. The recombinant non-replicating Ad expresses the Escherichia coli β-galactosidase (β-gal) reporter gene under control of the cytomegalovirus immediate-early enhancer region. We have also used methylene blue plus visible light (MB + VL) to induce the major oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG) in the recombinant Ad-encoded reporter gene in order to study base excision repair (BER). The reported variability regarding 8-oxoG's potential to block transcription by RNA polymerase II and data demonstrating that a number of factors play a role in transcriptional bypass of the lesion led us to examine the repair of 8-oxoG in the Ad reporter and its relationship to HCR for expression of the reporter gene. We have used Southern blotting to examine removal of UVC- and MB + VL-induced DNA damage by loss of endonuclease-sensitive sites from the Ad-encoded β-gal reporter gene in human and rodent cells. We show that repair of MB + VL-induced 8-oxoG via BER and UVC-induced cyclobutane pyrimidine dimers (CPDs) via NER is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. We also show that HCR for expression of the MB + VL-damaged and the UVC-damaged reporter gene is substantially greater in human SV40-transformed GM637F skin fibroblasts compared to hamster CHO-AA8 cells. The difference between the human and rodent cells in the removal of both 8-oxoG and CPDs from the damaged reporter gene was comparable to the difference in HCR for expression of the damaged reporter gene. These results suggest that the major factor for HCR of the MB + VL-treated reporter gene in mammalian cells is DNA repair in the Ad rather than lesion bypass.
authors.affiliation: Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario L8S 4K1, Canada.
authors.forename: Derrik M
authors.initials: DM
authors.lastname: Leach
authors.forename: Natalie J
authors.initials: NJ
authors.lastname: Zacal
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Rainbow
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015139:Blotting, Southern; D016466:CHO Cells; D003412:Cricetulus; D004249:DNA Damage / Q000528:radiation effects; D045647:DNA Glycosylases / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D043382:DNA-Formamidopyrimidine Glycosylase / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000473:pathology / Q000821:virology; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006147:Guanine / Q000031:analogs & derivatives; D054884:Host-Pathogen Interactions; D006801:Humans; D008027:Light; D011740:Pyrimidine Dimers / Q000235:genetics* / Q000378:metabolism; D014466:Ultraviolet Rays; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: C453560:7,8-dihydro-8-oxoguanine; D029968:Escherichia coli Proteins; D011740:Pyrimidine Dimers; D006147:Guanine; D001616:beta-Galactosidase; D045647:DNA Glycosylases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli