Nucleic Acids Research, 1994, Vol. 22, No. 23 4890-4897
© 1994
MOLECULAR BIOLOGY |
Kinetic and equilibrium binding studies of the human papillomavirus type-16 transcription regulatory protein E2 interacting with core enhancer elements
Cancer Research Unit, Department of Biology, University of York Heslington, York YO1 5DD, UK
*To whom correspondence should be addressed
Received September 20, 1994. Accepted October 12, 1994.
The human papillomaviruses (HPVs) are a family of DNA viruses which cause benign tumours of the skin and mucosa that infrequently progress to malignant carcinoma. The E2 open reading frame of HPV is thought to encode a papillomavirus-specific transcription factor which also has a role in viral replication. The E2 proteins of all papillomaviruses studied to date have been shown to bind specifically to the common conserved sequence ACC(N)6GGT found at multiple locations in their genomes. In the case of HPV-16, a ‘high risk’ genital papillomavirus, the E2 protein is thought to negatively regulate expression of the major viral transforming genes E6 and E7, which have been directly implicated in the oncogenic process. However, little information exists concerning the relative or absolute affinities of the native HPV-16 protein for its palindromic recognition sequences; moreover, interpretation of any transcription or replication phenomena attributed to this protein is more complicated in the absence of such data. Here we describe the over-expression, purification and characterisation of the C-terminal 89 amino acids of the protein encompassing the DNA binding/dimerisation domain. We show that the recombinant protein purified from E.coli by a combination of non-group-specific chromatography steps retains high biological activity and is able to bind to all sites in the HPV-16 genome with high affinity ({small tilde}8x10-11 M). In addition, kinetic studies show that the E2-DNA complexes are very stable, with half-lives ranging from 2.15 to greater than 240 min, and that nucleotides internal and external to the conserved palindrome appear to influence stability.
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