Conserved cut repeats in the human cut homeodomain protein function as DNA binding domains.
Homeodomain-containing proteins are believed to function as sequence-specific DNA binding proteins, regulating gene expression. Specificity of sequence recognition is conferred by the homeodomain acting either alone or in conjunction with other conserved DNA binding domains as is the case for Pou domain and Paired domain proteins. The recent isolation of cDNAs encoding mammalian homologues of the Drosophila Cut homeodomain protein has revealed that the 72-amino acid Cut Repeats are conserved in evolution. We have investigated the biochemical activity of human Cut Repeats by expressing fusion proteins containing glutathione S-transferase linked to various combinations of Cut Repeats and Cut homeodomain. We show by gel retardation and DNase footprinting assays that Cut Repeats can function as DNA binding domains, either independently or in cooperation with the homeodomain. The binding affinity (KD) to a specific recognition site was estimated to be 8 x 10(-9) M for Cut Repeat 3 and 4 x 10(-10) M for Cut Repeat 1. When both Cut Repeat 3 and the Cut homeodomain were present in the fusion protein, the binding affinity was increased to 4 x 10(-11) M. These results define a novel class of proteins that contain in addition to the homeodomain a second conserved protein domain, the Cut Repeats, that also function as a DNA binding domain.
Harada
R
R
Department of Medicine, McGill University, Royal Victoria Hospital, Montreal, Québec, Canada.
Dufort
D
D
Denis-Larose
C
C
Nepveu
A
A
D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003851:Deoxyribonucleases; D004330:Drosophila / Q000235:genetics / Q000378:metabolism; D005801:Genes, Homeobox; D005982:Glutathione Transferase / Q000378:metabolism; D018398:Homeodomain Proteins; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012097:Repressor Proteins; D014157:Transcription Factors
C080632:CUX1 protein, human; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase; D003851:Deoxyribonucleases