Orientation of a Novel DNA Binding Site Affects Human Papillomavirus-Mediated Transcription and Replication

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American Society for Microbiology

RESUMO

A consensus binding site for the human papillomavirus (HPV) E2 protein was determined from an unbiased set of degenerate oligonucleotides using cyclic amplification and selection of targets (CASTing). Detectable DNA-protein complexes were formed after six to nine cycles of CASTing. A population of selected binding sites was cloned, and a consensus was determined by statistical analysis of the DNA sequences of individual isolates. Starting from a pool with 20 random bases, a consensus binding site of ACAC-N5-GGT was derived. CASTing and electrophoretic mobility shift analyses demonstrate that human but not bovine papillomavirus E2 proteins recognize this sequence. The presence of this sequence in papillomavirus genomes suggests a role for its function. We demonstrate that this site functionally substitutes for the canonical E2 binding site (ACCG-N4-CGGT) in both transient-transcription and DNA replication assays. This sequence, in most instances, is interchangeable with the resident E2 binding sites in the context of the HPV type 16 long control region. Where the novel sequence does not support E2-mediated effects on gene expression or DNA replication, we demonstrate that changing the orientation of the novel sequence restores this effect.

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