Mobility of murine replication proteins in the nucleus

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Our long-term aim is the elucidation of the mechanisms governing the onset of DNA-replication at the G1/S-phase transition. The project will be focused on the dynamics of the initiator proteins during the assembly and activation of the pre-replicative complex. We will study the kinetic properties of individual initiator proteins in the nuclei of living cells with photobleaching techniques. Vectors encoding fusions of green fluorescence protein (GFP) with initiator proteins, e.g. ORC1-6, MCM2-7, CDC6, CDC45, DBF4, CDC7, PLK, CDT1 and geminin proteins will be used to measure the dynamics of replication proteins. We will apply FRAP (Fluorescence Recovery After Photobleaching) and FLIP (Fluorescence Loss In Photobleaching). The effect of knocking-out the expression of individual proteins on the assembly and localization of the pre-replicative complex will be investigated by RNA-interference. Finally, the effect of posttranslational modifications (phosphorylation, acetylation) of initiator proteins on the structural and functional dynamics of the replication machinery will be investigated.