A bovine papillomavirus E1-related protein binds specifically to bovine papillomavirus DNA.

The E1 open reading frame of bovine papillomavirus (BPV) was expressed as a RecA-E1 fusion protein in Escherichia coli. The bacterially expressed RecA-E1 protein exhibited sequence-specific DNA binding activity; strong binding to the region from nucleotides 7819 to 93 on the BPV genome (designated region A) and weak binding to the adjacent region from nucleotides 7457 to 7818 (region B) were observed. The interaction between the BPV-derived RecA-E1 protein and region A appeared to be highly specific for BPV DNA, as no comparable binding was detected with heterologous papillomavirus DNAs. Binding to region A was eliminated by digestion of region A at the unique HpaI site, which suggests that the RecA-E1 binding site(s) was at or near the HpaI recognition sequence. Binding to region B but not region A was observed when nuclear extracts from ID13 cells were used as a source of E1 proteins. The absence of region A binding by ID13 extracts may reflect a negative regulation of E1 DNA binding activity.     

Specificity in DNA recognition by a peptide from papillomavirus E2 protein

The E2 proteins of papillomavirus specifically bind to double-stranded DNA containing the consensus sequence ACCG-N4-CGGT, where N is any nucleotide. Here, we show the binding and recognition of dissimilar DNA sequences by an 18 amino-acid peptide (alpha1E2), which corresponds to the DNA-recognition helix, alpha-helix-1. Isothermal DNA binding assays performed with the DNA consensus sequence show saturable curves with alpha1E2 peptide, and the alpha1E2 peptide is converted to an ordered conformation upon complexation. Measurements performed with non-specific DNA sequence fail to saturate, a behavior characteristic of non-specific binding. Binding of the alpha1E2 peptide to these DNA sequences display a different counter-ion dependence, indicating a dissimilar, sequence-dependent mechanism of interaction. Quantitative stoichiometric measurements revealed the specificity in alpha1E2 peptide recognition of the ACCG half-site, demonstrating capacity for discrimination of nucleic acid bases sequences without the need of a whole protein architecture.     

Cooperative binding of the E2 protein of bovine papillomavirus to adjacent E2-responsive sequences.

The DNA-binding properties of purified full-length E2 protein from bovine papillomavirus type 1 have been investigated by utilizing a quantitative gel shift analysis. By using a recombinant baculovirus which express the E2 open reading frame from the polyhedrin promoter, the full-length E2 protein was synthesized in insect cells and purified to homogeneity by using an E2 binding site (ACCGN4CGGT)-specific oligonucleotide column. The Kd of E2 binding to a 41-bp oligonucleotide containing a single binding site was found to be 2 x 10(-11) M. When two binding sites were included on an oligonucleotide, cooperative binding to these sites by the E2 protein was observed. A cooperativity parameter of 8.5 was determined for E2 binding to two sites. An 86-amino-acid peptide encompassing the C terminus of the protein retains the ability to bind E2 binding sites with a Kd of 4 x 10(-10) M but exhibits slight cooperativity of binding to two adjacent sites. A major determinant for cooperative binding of the full-length E2 protein is thus encoded by the N-terminal amino acids outside the minimal DNA binding domain.     

Identification of a short, hydrophilic amino acid sequence critical for origin recognition by the bovine papillomavirus E1 protein

The E1 protein of bovine papillomavirus (BPV) is a site-specific DNA binding protein that recognizes an 18-bp inverted repeat element in the viral origin of replication. Sequence-specific DNA binding function maps to the region from approximately amino acids 140 to 300, and isolated polypeptides containing this region have been shown to retain origin binding in vitro. To investigate the sequence and structural characteristics which contribute to sequence-specific binding, the primary sequence of this region was examined for conserved features. The BPV E1 DNA binding domain (E1DBD) contains three major hydrophilic domains (HR1, amino acids 179-191; HR2, amino acids 218 to 230; and HR3, amino acids 241 to 252), of which only HR1 and HR3 are conserved among papillomavirus E1 proteins. E1DBD proteins with lysine-to-alanine mutations in HR1 and HR3 were severely impaired for DNA binding function in vitro, while a lysine-to-alanine mutation in HR2 had a minimal effect on DNA binding. Mutation of adjacent threonine residues in HR1 (T187 and T188) revealed that these two amino acids made drastically different contributions to DNA binding, with the T187 mutant being severely defective for origin binding whereas the T188 mutant was only mildly affected. Helical wheel projections of HR1 predict that T187 is on the same helical face as the critical lysine residues whereas T188 is on the opposing face, which is consistent with their respective contributions to DNA binding activity. To examine E1 binding in vivo, a yeast one-hybrid system was developed. Both full-length E1 and the E1DBD polypeptide were capable of specifically interacting with the E1 binding site in the context of the yeast genome, and HR1 was also critical for this in vivo interaction. Overall, our results indicate that HR1 is essential for origin binding by E1, and the features and properties of HR1 suggest that it may be part of a recognition sequence that mediates specific E1-nucleotide contacts.     

Protein flexibility directs DNA recognition by the papillomavirus E2 proteins

Although DNA flexibility is known to play an important role in DNA-protein interactions, the importance of protein flexibility is less well understood. Here, we show that protein dynamics are important in DNA recognition using the well-characterized human papillomavirus (HPV) type 6 E2 protein as a model system. We have compared the DNA binding properties of the HPV 6 E2 DNA binding domain (DBD) and a mutant lacking two C-terminal leucine residues that form part of the hydrophobic core of the protein. Deletion of these residues results in increased specific and non-specific DNA binding and an overall decrease in DNA binding specificity. Using (15)N NMR relaxation and hydrogen/deuterium exchange, we demonstrate that the mutation results in increased flexibility within the hydrophobic core and loop regions that orient the DNA binding helices. Stopped-flow kinetic studies indicate that increased flexibility alters DNA binding by increasing initial interactions with DNA but has little or no effect on the structural rearrangements that follow this step. Taken together these data demonstrate that subtle changes in protein dynamics have a major influence on protein-DNA interactions.     

Nonsense mutation in open reading frame E2 of bovine papillomavirus DNA.

Oligonucleotide-directed mutagenesis was used to construct a nonsense mutation in open reading frame (ORF) E2 of bovine papillomavirus DNA. A single base substitution mutation was constructed which converted a TAC codon into a TAG amber stop codon at a position in the ORF that did not overlap with any other viral ORFs. Full-length viral DNA containing the mutation induced only approximately 2% of the transformed foci of mouse C127 cells that were induced by wild-type DNA. In a different transformation assay, approximately one-half of the C127 cells which had acquired the mutant DNA gave rise to colonies containing at least some cells with transformed morphology. The constructed mutation was maintained in cell lines derived from cells which had acquired the mutant viral DNA, but the viral DNA appeared to be integrated into the host cell genome. Genetic mapping experiments proved that the constructed amber mutation caused the decrease in focus-forming activity and the integration of the mutant viral DNA. These results suggest that ORF E2 encodes a protein which is involved either directly or indirectly in some aspects of oncogenic transformation by bovine papillomavirus and in maintaining the viral DNA as a plasmid in transformed cells.     

CpG methylation directly inhibits binding of the human papillomavirus type 16 E2 protein to specific DNA sequences.

CpG methylation of the human papillomavirus upstream regulatory region has previously been shown to reduce virus promoter activity. Here, we demonstrate that methylation of the CpG dinucleotides contained within the binding site of the human papillomavirus type 16 E2 protein has a direct effect on the interaction of this protein with DNA. Methylation of both CpG dinucleotides within the E2 site abolishes the binding of E2.