The focus of this proposal is to explore oncogene control of mammalian DNA replication. We propose to use polyomavirus middle T-antigen (MT) as a model oncogene and the Py origin as a model of a eukaryotic origin. The rationale is based upon our recent discovery that MT plays a crucial role in Py DNA replication. This MT role is mediated by factors which are presumably activated as downstream targets of the MT signal transduction and which bind in the enhancer upstream of the viral origin. The most likely factor candidates are PEA1 and PEA3, the mouse homologs of AP1 and c-ets. The bulk of the MT effect appears to take place at the level of origin firing, converting the replication of the viral genome to an uncontrolled state. To understand MT stimulation, we will map cis-acting sequences in the Py enhancer (MTRE) which can stimulate DNA replication from the Py origin in response to MT and will examine their relative role in transcription and replication; we will sort out the relative contributions of MT, small T and specific MT domains towards stimulation of replication; we will examine the level(s) at which factor activation is effected (transcript levels, protein level, protein modification, protein stability, DNA binding) and relate these to specific domains of MT. In the second part, we will begin to analyze the impact of MT on the timing, rate and number of rounds of replication through the S-phase. The phenomena studied are tied with central issues in the regulation of cell growth in normal and oncogenic conditions, namely the control of origin firing. The potential outcome of the experiments is the finding that an important aspect of oncogenic deregulation takes place at the level of the control origin firing, perhaps by altering the licensing factor, and that oncogenically activated transcription factors play a key in this process.
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