Organization of the cis-acting element required for wheat dwarf geminivirus DNA replication and visualization of a rep protein-DNA complex.

摘要

The DNA sequences present in the large intergenic region (LIR) of wheat dwarf monogeminivirus (WDV), a subgroup I member of the geminivirus family, which are required for viral DNA replication were analysed. The boundaries of the viral cis-acting DNA replication element were defined, the contribution of different domains of the LIR to DNA replication efficiency determined and WDV Rep-DNA complexes visualized. Analysis of unidirectional deletions from both sides of the LIR established that a c. 200-bp cis-acting element (core) is essential for viral DNA replication. It spans c. 170 and 28 bp upstream and downstream, respectively, from the initiation site (+1), located in the invariant loop. This core element is flanked, at each side, by auxiliary regions (5'-aux and 3'-aux, c. 70 and c. 25 bp long, respectively), which contain DNA sequences that simulate DNA replication. Competition experiments using viral replicating vectors bearing wild-type or mutant WDV LIRs suggest that the auxiliary regionsmay contribute to the stabilization and/or activity of the initiation complex formed by WDV Rep at the origin. DNA-protein complexes were visualized by electron microscopy and a high-affinity binding site of WDV Rep protein within the core element has been mapped to c. 144 ± 18 bp upstream from the initiation site, between the start for complementary-sense transcription and the TATA box. It is concluded that the modular structure of the WDV DNA replication cis-acting element is established and direct evidence for the formation in vitro of a large nucleoprotein complex within the essential cis-acting element is provided.