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DNA binding and bending by the human papillomavirus type 16 E2 protein. Recognition of an extended binding site
1Department of Biochemistry, Centre for Molecular Recognition, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
Abstract:
The human papillomavirus (HPV) 16 E2 protein (hE2) binds to four sites present upstream of the P97 promoter and regulates transcription of the viral E6 and E7 oncogenes. We have determined the relative binding constants for the interaction of the full-length hE2 protein with these sites. Our results show that hE2 binds tightly to site 4, less tightly to sites 1 and 2, and weakly to site 3. Similar results have previously been obtained using a C-terminal fragment of the hE2 protein suggesting that the C-terminal domain is the sole determinant of DNA binding affinity and specificity. Using circular permutation assays we show that binding of the hE2 protein induces the formation of a significant DNA bend and that the hE2-induced DNA bend angle is the same at both tight and weak hE2-binding sites. An alignment of the four hE2-binding sites from the HPV 16 genome suggests that this protein recognizes an extended binding site when compared with the bovine papillomavirus E2 protein. Here we show that the hE2 protein binds tightly to sites containing an A:T or a G:C base pair at position 7 of its binding site but weakly to sites containing either C:G or T:A at this position. Using site-directed mutagenesis we demonstrate that an arginine at position 304 of the hE2 protein is responsible for the recognition of specific base pairs at this position.
Insights
Human papillomavirus (HPV) 16 E2 protein binding to DNA sites regulates oncogene transcription. The C-terminal domain determines binding affinity, and specific arginine residues recognize DNA base pairs.
Area of Science:
- Molecular Biology
- Virology
- Structural Biology
Background:
- Human papillomavirus (HPV) 16 E2 protein (hE2) regulates viral oncogene transcription.
- hE2 binds to specific DNA sites upstream of the P97 promoter, controlling E6 and E7 oncogene expression.
Purpose of the Study:
- To determine the relative binding affinities of the full-length hE2 protein to its four DNA binding sites.
- To investigate the role of the C-terminal domain in hE2 DNA binding specificity.
- To characterize the DNA bending induced by hE2 binding and identify key residues for base-pair recognition.
Main Methods:
- Determination of relative binding constants for hE2 protein interaction with DNA sites.
- Circular permutation assays to assess DNA bending upon hE2 binding.
- Site-directed mutagenesis to identify amino acid residues critical for DNA binding specificity.
Main Results:
- hE2 exhibits differential binding affinities, with tightest binding to site 4, intermediate to sites 1 and 2, and weakest to site 3.
- The C-terminal domain of hE2 appears to be the primary determinant of DNA binding affinity and specificity.
- hE2 binding induces a significant DNA bend, with a consistent bend angle across different binding sites.
- hE2 preferentially binds sites with A:T or G:C at position 7, but weakly binds sites with C:G or T:A at this position.
- Arginine at position 304 of hE2 is crucial for recognizing specific base pairs at position 7.
Conclusions:
- The C-terminal domain of HPV 16 E2 protein dictates DNA binding affinity and specificity.
- hE2 binding induces DNA bending, a conserved feature across different binding affinities.
- Specific base pair recognition at position 7 of the binding site is mediated by arginine 304, influencing differential binding strengths.