The DNA-binding domain of HPV-16 E2 protein interaction with the viral enhancer: protein-induced DNA bending and role of the nonconserved core sequence in binding site affinity.
We expressed the carboxy-terminal portion of the E2 open reading frame (ORF)-encoded protein of human papillomavirus type 16 (HPV-16) and purified it to near homogeneity. Using DNase I footprinting techniques, we show that like the homologous protein from bovine papillomavirus type 1 (BPV-1), HPV-18, HPV-11, it binds DNA at the enhancer consensus motif ACCN6GGT. Base and phosphate backbone contact points were determined using methylation protection and interference and ethylation interference assays. This HPV-16 E2 DNA-binding domain protein contacts the site at the outermost conserved GG residues which is similar to the interaction of the BPV-1 E2 system. However, there are many fewer phosphate backbone contacts. Using gel retardation assays, the HPV-16 E2 protein interaction with the consensus motif was characterized further based on the specific sequence of the noncontacted, nonconserved internal bases. Affinity of this E2 protein for the consensus site increased dramatically with an A.T-rich core sequence. Like the homologous BPV-1 protein, HPV-16 E2 protein induces DNA bending at its binding site. Furthermore, examination of the DNA region containing a single consensus motif far upstream from the major promoter, P97, revealed naturally bent DNA that was further bent upon interaction with the HPV-16 E2 protein.
Bedrosian
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Division of Hematology and Oncology, Duke University Medical Center, Durham, North Carolina 27710.
Bastia
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D000478:Alkylation; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000032:analysis / Q000378:metabolism*; D027383:Papillomaviridae
D004268:DNA-Binding Proteins; C054855:E2 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D004247:DNA