RESUMEN
The Geminiviridae family includes a large number of viruses that infect plants and have a unique geminate virion particle, a single-stranded genome of approximately 2.6-3.0 kb, and replicate through a rolling-circle mechanism. Since they encode for just a few proteins (4-6 depending on the members that belong to four different genera), a rich variety of interactions has evolved between viral proteins and host factors to develop the virus replicative cycle. Among them, we have been particularly interested so far: (i). in the interference with cell cycle regulatory proteins of the retinoblastoma-related (RBR)/E2F pathway and (ii). in the interaction with host DNA replication factors necessary for the assembly of a functional replication complex at the viral origin of DNA replication during the rolling-circle stage. Yeast two-hybrid assays revealed that wheat dwarf virus RepA protein, but nor Rep protein, interacts with plant RBR protein. Interestingly, deletion of the C-terminal domain of Rep confers the truncated protein the ability to interact with RBR, suggesting that this domain may hinder the LXCXE RBR-binding motif. Secondary structure predictions support such a possibility.
Asunto(s)
Replicación del ADN/fisiología , Geminiviridae/fisiología , Genoma Viral , Replicación Viral/genética , Secuencia de Aminoácidos , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/metabolismo , Geminiviridae/genética , Geminiviridae/crecimiento & desarrollo , Geminiviridae/metabolismo , Datos de Secuencia Molecular , Origen de Réplica , Proteína de Retinoblastoma/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Genetic evidence suggests that the SPP1-encoded gene 35 product (G35P) is essential for phage DNA replication. Purified G35P binds single-strand DNA (ssDNA) and double-strand (dsDNA) and specifically interacts with SPP1-encoded replicative DNA helicase G40P and SSB protein G36P. G35P promotes joint molecule formation between a circular ssDNA and a homologous linear dsDNA with an ssDNA tail. Joint molecule formation requires a metal ion but is independent of a nucleotide cofactor. Joint molecules formed during these reactions contain a displaced linear ssDNA strand. Electron microscopic analysis shows that G35P forms a multimeric ring structure in ssDNA tails of dsDNA molecules and left-handed filaments on ssDNA. G35P promotes strand annealing at the AT-rich region of SPP1 oriL on a supercoiled template. These results altogether are consistent with the hypothesis that the homologous pairing catalyzed by G35P is an integral part of SPP1 DNA replication. The loading of G40P at a d-loop (ori DNA or at any stalled replication fork) by G35P could lead to replication fork reactivation.