In plants and animals the E2F-RB pathway is one the most important cellular mechanisms that control the decision to continue or stop cell division. E2Fs are a family of proteins that require the association with the DP proteins for control the transcription of genes involved in cell cycle transitions and DNA synthesis, e.g. components of the pre-replication complexes (pre-RC), among others. The function of these E2F/DP factors is negatively regulated by the retinoblastoma protein (RB), which binds to E2F proteins and block their trans-activating potential. Arabidopsis, has six E2Fs (E2Fa through E2Ff) and 2DPs (DPa and DPb) genes. Two of them, E2Fa and E2Fc have been analysed in more detail: E2Fa acts as a transcriptional activator while E2Fc as a repressor.
Recent studies in Drosophila and mammalian cells have revealed a novel function of the E2F/RB pathway. This is its involvement in regulating directly the activity of DNA replication origins. Thus, a RB-mediated association between the E2F/RB complex and certain O RC proteins has been observed. The E2F-RB-ORC complex seems to limit DNA replication by inhibiting the activation of DNA replication origins.
The present project is specifically aimed at elucidating the potential implications of the plant E2F/RB pathway in regulating ORC function and, eventually, DNA replication origin activity. To this end, we will use Arabidopsis as a model system. In a first step, we will generate a full interaction map of all six Arabidopsis E2Fs with each Arabidopsis ORC subunit using a yeast two-hybrid approach; we will confirm positive interaction by in vitro pull-down experiments using purified fusion proteins expressed in bacteria. The receiving laboratory has generated a number of transgenic plants expressing tagged versions of proteins relevant for this project and antibodies against E2Fc and E2Ff are also available.
Fields of science
Call for proposal
See other projects for this call