Transcription of baculovirus late genes is absolutely dependent upon viral DNA replication, which is believed to create or unmask a cis-acting signal in the DNA template. In vitro analyses with the viral-encoded RNA polymerase suggests that this signal is the replication fork, and that proteins bound to the fork enhance late transcription. The goals of this proposal are to identify the proteins that enhance late transcription and to define their mechanism of action. Enhancers will be purified using a complementation assay, which consists of purified viral RNA polymerase, a DNA template containing a replication fork, and chromatography fractions. Forked templates are preferentially enhanced suggesting that proteins bind specifically to replication forks. Therefore, the DNA binding specificity of the enhancers will be determined by electrophoretic mobility shift assays using linear, nicked, and forked probes. The interactions between the enhancers, RNA polymerase, forks, and baculovirus promoters will be analyzed by protein cross-linking and DNase I footprinting assays. The mechanism of enhancer function will be further characterized by permanganate probing and abortive initiation assays. Finally, to determine whether the replication fork is a position and orientation-independent element, different circular and linear templates, which contain replication forks upstream, downstream, and on both strands of DNA will be constructed. Comparison of the transcription activities of these templates will help to determine whether the replication fork enhancers function similarly to classical transcription enhancers. These experiments should provide an explanation for the dependence of late transcription on DNA replication. The enhancer factors act preferentially on forked DNA templates, and the primary intracellular mechanism of generating forked templates is through DNA replication. If the viral RNA polymerase is dependent on forked structures for enhanced transcription, then late transcription cannot occur in the absence of DNA replication, even though RNA polymerase and the late transcription factors are encoded by early genes. Knowledge gained from these experiments should be relevant to other viral systems and will provide a better understanding of the complex interactions between transcription and replication machinery in eukaryotic cells.
