KINETIC AND EQUILIBRIUM BINDING-STUDIES OF THE HUMAN PAPILLOMAVIRUS TYPE-16 TRANSCRIPTION REGULATORY PROTEIN E2 INTERACTING WITH CORE ENHANCER ELEMENTS

The human papillomaviruses (HPVs) are a family of DNA viruses which ca use benign tumours of the skin and mucosa that infrequently progress t o 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 st udied to date have been shown to bind specifically to the common conse rved sequence ACC(N)(6)GGT found at multiple locations in their genome s. In the case of HPV-16, a 'high risk' genital papillomavirus, the E2 protein is thought to negatively regulate expression of the major vir al transforming genes E6 and E7, which have been directly implicated i n 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 ove rexpression, purification and characterisation of the C-terminal 89 am ino acids of the protein encompassing the DNA binding/dimerisation dom ain. We show that the recombinant protein purified from E.coli by a co mbination of non-group-specific chromatography steps retains high biol ogical activity and is able to bind to all sites in the HPV-16 genome with high affinity (similar to 8x1-11 M). In addition, kinetic studies show that the E2 - DNA complexes are very stable, with half-lives ran ging from 2.15 to greater than 240 min, and that nucleotides internal and external to the conserved palindrome appear to influence stability .