Organization of thecis-Acting Element Required for Wheat Dwarf Geminivirus DNA Replication and Visualization of a Rep Protein–DNA Complex

Abstract

Initiation of geminivirus DNA replication depends on the activity of the initiator protein (Rep) upon interaction with DNA sequences present in the intergenic region of the viral DNA. In this study, we have analyzed the DNA sequences present in the large intergenic region (LIR) of wheat dwarf virus (WDV), a subgroup I member of the geminivirus family, which are required for viral DNA replication. We have (i) defined the boundaries of the viralcis-acting DNA replication element, (ii) determined the contribution of different domains of the LIR to DNA replication efficiency, and (iii) visualized WDV Rep–DNA complexes. Analysis of unidirectional deletions from both sides of the LIR leads us to establish that a ∼200-bpcis-acting element (core) is essential for viral DNA replication. It spans ∼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, ∼70 and ∼25 bp long, respectively), which contain DNA sequences that stimulate DNA replication. Competition experiments using viral replicating vectors bearing wild-type or mutant WDV LIRs suggest that the auxiliary regions may contribute to the stabilization and/or activity of the initiation complex formed by WDV Rep at the origin. We have visualized DNA–protein complexes by electron microscopy and a high-affinity binding site of WDV Rep protein within the core element has been mapped to ∼144 ± 18 bp upstream from the initiation site, between the start site for complementary-sense transcription and the TATA box. Our studies (i) establish the modular structure of the WDV DNA replicationcis-acting element and (ii) provide direct evidence for the formationin vitroof a large nucleoprotein complex within the essentialcis-acting element.