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.
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