A fluorescence anisotropy study of DNA binding by HPV-11 E2C protein: a hierarchy of E2-binding sites.
Association of the human papillomavirus (HPV) E2 protein with its palindromic DNA-binding site is a necessary step for transcriptional trans-activation. To study the interaction between DNA and E2, the carboxyl-terminal domain of HPV-11 E2 protein (E2C) was expressed in Escherichia coli and purified to homogeneity. The binding affinity of the recombinant E2C protein for a single palindromic DNA recognition site was determined using a 5'-fluorescein-labeled 24 base pair oligonucleotide. Competitive titrations between the fluorescein-labeled oligonucleotide and an unlabeled oligonucleotide of identical sequence yielded a native affinity of 4.5 x 10(-9)M. Sequences from the seven E2-binding sites within the HPV-11 genome were titrated to establish a hierarchy of binding site affinities. All high-affinity E2-binding sites are located within or near the HPV-11 LCR. E2-binding sites distant from the LCR appear to have low affinity for E2. When the location and affinity of each E2-binding site are plotted in relation to a transcription map of HPV-11, it is apparent that the major RNA transcripts produced reflect the high-affinity E2-binding sites within the HPV LCR. To assess the E2C-binding contribution of specific base pairs within the oligonucleotide palindrome, additional double-stranded oligonucleotides were prepared in which the central nonpalindromic sequences were varied. While simple strand transposition of the A4.T4 center had a minimal effect upon the E2C-oligonucleotide binding affinity, replacement with TATA.ATAT or CGCG.GCGC centers substantially decreased the affinity of E2C for its binding site. Alteration of the canonical portions of the E2-binding palindrome reduced the DNA-protein binding affinity dramatically.
Alexander
K A
KA
Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, USA.
Phelps
W C
WC
D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods; D016679:Genome, Viral; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism*; D009856:Oncogene Proteins, Viral / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D027383:Papillomaviridae / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry / Q000378:metabolism
D017931:DNA Primers; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D009856:Oncogene Proteins, Viral; D011994:Recombinant Proteins; D015534:Trans-Activators; C076036:oncogene protein E2, Human papillomavirus type 1; D004247:DNA