DNA-binding properties of CCAAT displacement protein cut repeats.
CCAAT displacement protein (CDP) is a transcriptional repressor that contains four distinct DNA-binding domains; a homeodomain and three cut repeats. Each DNA-binding domain of CDP was expressed as a glutathione S-transferase (GST)-fusion protein and analyzed for relative binding affinity to five CDP-binding sites within the gp91phox promoter. Each cut repeat exhibits a unique pattern of DNA-binding affinities for the five binding sites in the gp91phox promoter, suggesting that each may make a distinct contribution to the DNA-binding behavior of native CDP. Although measurement of DNA/protein complex mass indicates that an isolated cut repeat can bind DNA as a monomer, mixing of GST-cut repeat and GST-homeodomain fusion proteins enhances DNA-binding activity. Far-Western blot and two-hybrid analyses indicate, however, that the CDP domains do not directly interact. We hypothesize that GST-mediated dimerization leads to spatial juxtaposition of these DNA-binding domains, and that the resulting enhanced DNA-binding activity mimics cooperative interactions that occur between these domains in native CDP.
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Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis 46202, USA.
Luo
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Skalnik
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D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005809:Genes, Regulator; D005982:Glutathione Transferase / Q000235:genetics; D018398:Homeodomain Proteins; D006801:Humans; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020080:Tandem Repeat Sequences; D014157:Transcription Factors; D015533:Transcriptional Activation
C080632:CUX1 protein, human; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D005982:Glutathione Transferase