Assessing Redundancy in DNA Replication Initiation (360G-Wellcome-206897_Z_17_Z)

CDC7 kinase is a key molecular switch for DNA replication. CDC7 kinase is a protein kinase which phosphorylates and activates the mini-chromosome maintenance (MCM) helicase thus triggering DNA replication origin firing. However, recent research has shown that CDC7 kinase inhibition with some ATP competitive inhibitors does not entirely inhibit DNA replication and cell cycle can still proceed, thus have marginal effects on cell proliferation. Importantly the FANCI protein, that is normally involved in DNA repair, has been shown to be involved for the firing of DNA replication origins during replication stress and to regulate CDC7 dependent activation of MCM helicase at "dormant origins" . We will test the hypothesis that CDC7 and FANCI cooperate in the activation origin during normal replication and that the activity of CDC7 inhibitors could be greatly enhanced in FANCI depleted cells. For this we will deplete FANCI from breast derived cells and traet them with different doses of the CDC7 inhibitor XL413. Key goals are 1) reproducible detection of FANCI by western blotting, 2) efficacious depletion of FANCI by siRNA and 3) implementation of DNA replication and cell survival assays.