Effect of oxidative DNA damage in promoter elements on transcription factor binding.
Reactive oxygen species produced by endogenous metabolic activity and exposure to a multitude of exogenous agents impact cells in a variety of ways. The DNA base damage 8-oxodeoxyguanosine (8-oxodG) is a prominent indicator of oxidative stress and has been well-characterized as a premutagenic lesion in mammalian cells and putative initiator of the carcinogenic process. Commensurate with the recent interest in epigenetic pathways of cancer causation we investigated how 8-oxodG alters the interaction between cis elements located on gene promoters and sequence-specific DNA binding proteins associated with these promoters. Consensus binding sequences for the transcription factors AP-1, NF-kappaB and Sp1 were modified site-specifically at guanine residues and electrophoretic mobility shift assays were performed to assess DNA-protein interactions. Our results indicate that whereas a single 8-oxodG was sufficient to inhibit transcription factor binding to AP-1 and Sp1 sequences it had no effect on binding to NF-kappaB, regardless of its position. We conclude from these data that minor alterations in base composition at a crucial position within some, but not all, promoter elements have the ability to disrupt transcription factor binding. The lack of inhibition by damaged NF-kappaB sequences suggests that DNA-protein contact sites may not be as determinative for stable p50 binding to this promoter as other, as yet undefined, structural parameters.
Ghosh
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Department of Carcinogenesis, University of Texas M. D. Anderson Cancer Center, Science Park/Research Division, Smithville, TX 78957, USA.
Mitchell
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DL
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics*; D018983:DNA Footprinting; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003848:Deoxyguanine Nucleotides / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D003848:Deoxyguanine Nucleotides; D016328:NF-kappa B; D016329:Sp1 Transcription Factor; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C078206:8-oxodeoxyguanosine triphosphate; D004247:DNA