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. Author manuscript; available in PMC: 2023 Jun 15.
Published in final edited form as: Mol Cell. 2023 Jan 5;83(1):26–42.e13. doi: 10.1016/j.molcel.2022.12.004

Figure 7. CDT1 inhibits CMG helicase through its MCM-binding domains.

Figure 7

(A) Mechanisms underlying inhibited replication fork progression.

(B) Mitogen-released MCF10A cells were treated with 2 mM hydroxyurea (HU) and 2 μM AZ-20 (ATRi) during the final 4 h. G1 and S cells were identified (see STAR Methods). n ≥ 182 cells per condition. Dashed line is G1 signal median. Representative of 3 independent experiments.

(C) Top: polymerase epsilon primer extension assay (M13mp18 circular single-stranded DNA template). Bottom: denaturing agarose gel analysis of 15 min primer extension reactions. 10 nM CDT1 pre-incubated with equimolar geminin where indicated. Representative of 3 independent experiments.

(D) CDC45-GFP U2OS cells with doxycycline (Dox-inducible ND-CDT1 were treated with Dox for 8 h, pre-extracted, and co-stained for ND-CDT1(anti-HA-tag) and CDC45 (anti-GFP). Mid- or late-S-phase cells identified by CDC45 pattern. Signals were masked by thresholding. Yellow box is zoomed region. Representative of 35 (mid S) or 18 (late S) cells. Scale bars, 5 μm (full image) and 1 μm (zoom). Co-localization analysis in Figure S7B.

(E) Left: MCM-binding region mutants of ND-CDT1. WT-CDT1, wild-type CDT1. Right: dose responses of EdU to ND-CDT1 (anti-HA-tag), 1–2 h after S entry in mitogen-released siGeminin-treated MCF10A cells. Error bars are mean ± 2 × SEM(n ≥ 26 cells per bin, n ≥ 1,048 cells per condition). Dashed lines are negative threshold. Mutant expression in Figure S7C.

(F) Diagram of CDT1 regulation of DNA synthesis in early S.

See also Figure S7.