The bovine papillomavirus E2 transactivator is stimulated by the E1 initiator through the E2 activation domain

AbstractBovine papillomavirus type 1 (BPV-1) encodes two regulatory proteins, E1 and E2, that are essential for viral replication and transcription. E1, an ATP-dependent helicase, binds to the viral ori and is essential for viral replication, while the viral transcriptional activator, E2, plays cis-dominant roles in both viral replication and transcription. At low reporter concentrations, E1 stimulates E2 enhancer function, while at high reporter concentrations, repression results. An analysis of cis requirements revealed that neither replication nor specific E1-binding sites are required for the initiators' effect on E2 transactivator function. Though no dependence on E1-binding sites was found, analysis of E1 DNA binding and ATPase mutants revealed that both domains are required for E1 modulation of E2. Through the use of E2 fusion-gene constructs we showed that a heterologous DNA-binding domain could be substituted for the E2 DNA-binding domain and this recombinant protein remained responsive to E1. Furthermore, E1 could rescue activation domain mutants of E2 defective for transactivation. These data suggest that E1 stimulation of E2 involves interactions between E1 and the E2 activation domain on DNA. We speculate that E1 may allosterically interact with the E2 activation domain, perhaps stabilizing a particular structure, which increases the enhancer function of E2

A Drosophila homolog of the yeast origin recognition complex

Genes from Drosophila melanogaster have been identified that encode proteins homologous to Orc2p and Orc5p of the Saccharomyces cerevisiae origin recognition complex (ORC). The abundance of the Drosophila Orc2p homolog DmORC2 is developmentally regulated and is greatest during the earliest stages of embryogenesis, concomitant with the highest rate of DNA replication. Fractionation of embryo nuclear extracts revealed that DmORC2 is found in a tightly associated complex with five additional polypeptides, much like the yeast ORC. These studies will enable direct testing of the initiator-based model of replication in a metazoan

Separation of origin recognition complex functions by cross-species complementation

Transcriptional silencing at the HMRa locus of Saccharomyces cerevisiae requires the function of the origin recognition complex (ORC), the replication initiator of yeast. Expression of a Drosophila melanogaster Orc2 complementary DNA in the yeast orc2-1 strain, which is defective for replication and silencing, complemented the silencing defect but not the replication defect; this result indicated that the replication and silencing functions of ORC were separable. The orc2-1 mutation mapped to the region of greatest homology between the Drosophila and yeast proteins. The silent state mediated by DmOrc2 was epigenetic; it was propagated during mitotic divisions in a relatively stable way, whereas the nonsilent state was metastable. In contrast, the silent state was erased during meiosis