Figure 2: Cdt1 licensing activity is negatively regulated by its PEST domain in vivo. | Nature Communications

Figure 2: Cdt1 licensing activity is negatively regulated by its PEST domain in vivo.

From: A spontaneous Cdt1 mutation in 129 mouse strains reveals a regulatory domain restraining replication licensing

Figure 2

(a) Constructs used in this study. (b) Re-replication assays showing the enhanced licensing activity of the Cdt1 PEST domain deletion mutants. U20S cells were cotransfected with the indicated mouse Cdt1 variants (mCdt1) and EGFP at the indicated ratios. Sixty hours post transfection, cells were processed and their DNA content analysed by flow cytometry. Transfected cells were gated based on the GFP signal. The percentage of re-replicating cells (>4n) is indicated. See Supplementary Figure S2 for further details. (c) Expression of the Cdt1 variants in the U2OS cells analysed in b was detected by immunoblotting. (d) Deletion of the PEST domain stimulates Cdt1 licensing activity in vivo. U20S cells were cotransfected with Flag-tagged MCM2 and MCM5, and Cdt1 variants (WT or ΔDDPEST), or empty vector (Vect) to monitor licensing in transfected cells exclusively. Protein levels in Triton-soluble and chromatin fractions were analysed by immunoblotting. (e) The regulatory role of the Cdt1 PEST domain is conserved. Re-replication assays of WT and ΔPEST human Cdt1 (hCdt1) were performed as in b. The percentage of re-replicating cells (>4n) is indicated. (f) Expression of Cdt1 corresponding to the experiment described in e.

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