title: CCAAT displacement activity involves CUT repeats 1 and 2, not the CUT homeodomain.
abstract: The CCAAT displacement protein, the homolog of the Drosophila melanogaster CUT protein, contains four DNA-binding domains: three CUT repeats (CR1, CR2, and CR3) and the CUT homeodomain (HD). Using a panel of fusion proteins, we found that a CUT repeat cannot bind to DNA as a monomer, but that certain combinations of domains exhibit high DNA-binding affinity: CR1+2, CR3HD, CR1HD, and CR2HD. One combination (CR1+2) exhibited strikingly different DNA-binding kinetics and specificities. CR1+2 displayed rapid on and off rates and bound preferably to two C(A/G)AT sites, organized as direct or inverted repeats. Accordingly, only CR1+2 was able to bind to the CCAAT sequence, and its affinity was increased by the presence of a C(A/G)AT site at close proximity. A purified CCAAT displacement protein/CUT protein exhibited DNA-binding properties similar to those of CR1+2; and in nuclear extracts, the CCAAT displacement activity also required the simultaneous presence of a C(A/G)AT site. Moreover, CR1+2, but not CR3HD, was able to displace nuclear factor Y. Thus, the CCAAT displacement activity requires the presence of an additional sequence (CAAT or CGAT) and involves CR1 and CR2, but not the CUT homeodomain.
authors.affiliation: Molecular Oncology Group, McGill University Health Center, and the Departments of Biochemistry, Medicine, and Oncology, McGill University, Montreal, Quebec H3A 1A1, Canada.
authors.forename: N S
authors.initials: NS
authors.lastname: Moon
authors.forename: G
authors.initials: G
authors.lastname: Bérubé
authors.forename: A
authors.initials: A
authors.lastname: Nepveu
mesh_terms: D000818:Animals; D001665:Binding Sites; D023081:CCAAT-Binding Factor / Q000737:chemistry / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D019175:DNA Methylation; D017931:DNA Primers / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004330:Drosophila; D029721:Drosophila Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D018398:Homeodomain Proteins; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics*; D014157:Transcription Factors
chemical_list: D023081:CCAAT-Binding Factor; C080632:CUX1 protein, human; D017931:DNA Primers; D018076:DNA, Complementary; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C066381:ct protein, Drosophila; D004247:DNA