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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
- Source :
- DNA repair. 1(11)
- Publication Year :
- 2003
-
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.
- Subjects :
- Xeroderma pigmentosum
DNA Repair
Transcription, Genetic
DNA repair
TATA box
CAAT box
Cytomegalovirus
Electrophoretic Mobility Shift Assay
Biology
Transfection
Biochemistry
medicine
Humans
Luciferases
Promoter Regions, Genetic
Molecular Biology
Cells, Cultured
DNA Primers
Xeroderma Pigmentosum
Deoxyadenosines
TAF9
Cell Biology
Base excision repair
medicine.disease
TATA-Box Binding Protein
Molecular biology
TATA Box
Oxidative Stress
Gene Expression Regulation
Mutation
biology.protein
Mutagenesis, Site-Directed
TATA-binding protein
Nucleotide excision repair
DNA Damage
Plasmids
Subjects
Details
- ISSN :
- 15687864
- Volume :
- 1
- Issue :
- 11
- Database :
- OpenAIRE
- Journal :
- DNA repair
- Accession number :
- edsair.doi.dedup.....ba070474c0887aff5ec4556436318824