title: A single 8,5'-cyclo-2'-deoxyadenosine lesion in a TATA box prevents binding of the TATA binding protein and strongly reduces transcription in vivo.
abstract: 8,5'-Cyclo-2'-deoxypurine (cPu) lesions result from the action of the hydroxyl radical on DNA. These lesions represent a unique class of oxidative DNA lesions in that they are repaired by the nucleotide excision repair (NER) pathway but not by base excision repair (BER) or direct repair. Previous work has shown that cyclopurines can block mammalian DNA and RNA polymerases. Thus, these lesions are of interest because of their potential role in the neurodegeneration as well as internal cancers observed in patients with xeroderma pigmentosum (XP) who lack the capacity to carry out NER. In the present work, we found that the S-isomer of 8,5'-cyclo-2'-deoxyadenosine (cA) can prevent binding of the TATA binding protein (TBP) to the TATA box from the CMV promoter. To assess the functional importance of this effect in living cells, we transfected constructs containing a single cA in the CMV TATA box into XP cells to determine the effect of the lesion on gene expression in vivo. Using this approach, we found that the lesion reduced gene expression by approximately 75%. This effect was comparable to the effect of an inactivating mutation of the TATA box in the same promoter. These findings identify an additional biological effect of cyclopurine lesions in mammalian cells, which is the ability to interfere with transcription by preventing transcription factor binding to cognate recognition sequences. In addition, the approach we used in this study represents a novel method for assessing the effects of DNA lesions in non-transcribed sequences on gene expression in living cells.
authors.affiliation: Section on Molecular Neurobiology, Laboratory of Neurogenetics, National Institute on Alcohol Abuse and Alcoholism, NIH, Bethesda, MD 20892-8110, USA.
authors.forename: Cheryl
authors.initials: C
authors.lastname: Marietta
authors.forename: Huzaefah
authors.initials: H
authors.lastname: Gulam
authors.forename: P J
authors.initials: PJ
authors.lastname: Brooks
mesh_terms: D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects; D017931:DNA Primers; D004260:DNA Repair / Q000187:drug effects*; D003839:Deoxyadenosines / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D018384:Oxidative Stress; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics; D016385:TATA Box / Q000235:genetics*; D035181:TATA-Box Binding Protein / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014983:Xeroderma Pigmentosum / Q000235:genetics* / Q000378:metabolism
chemical_list: D017931:DNA Primers; D003839:Deoxyadenosines; D035181:TATA-Box Binding Protein; C056752:8,5'-cyclo-2'-deoxyadenosine; D008156:Luciferases