Fig. 6: DONSON stabilizes the CMG helicase by suppressing precocious loading of the Cul2-LRR1 complex onto the CMG helicase. | Nature Communications

Fig. 6: DONSON stabilizes the CMG helicase by suppressing precocious loading of the Cul2-LRR1 complex onto the CMG helicase.

From: The DNA replication machinery transmits dual signals to prevent unscheduled licensing and execution of centrosome duplication

Fig. 6

a The Predicted Aligned Error (PAE) plot of the AlphaFold-Multimer (AF-M)-predicted DONSON-MCM3 and DONSON-MCM6 complex structure. b Ribbon representation of the predicted DONSON-MCM3 complex structure corresponding to (a). c HEK293T cells co-expressing MCM3-3×FLAG and HA-DONSON or the indicated deletion mutant were immunoprecipitated with FLAG antibodies. d A schematic of MCM7 poly-ubiquitination mediated by Cul2-LRR1 E3-ubiquitin ligase based on the previous studies. e The Predicted Aligned Error (PAE) plot of the AlphaFold-Multimer (AF-M)-predicted complex structure of MCM3/5/7-LRR1-ELOB-ELOC-Cul2-RBX1 with or without DONSON. f Representative structural models of the predicted protein complexes corresponding to the PAE plots in (e). All proteins are indicated in different colors. In the protein complex on the right, which lacks DONSON, the primary ubiquitination sites of MCM7, K26 and K27 (light blue), can be in close proximity (approximately 50 Å) to the C-terminal region of Cul2 and RBX1. g The interaction between endogenous MCM3 and LRR1 upon DONSON depletion. HeLa cells were treated with siControl or siDONSON for 48 h, and endogenous MCM3 was immunoprecipitated with an antibody against MCM3. h The amount of chromatin-bound LRR1 in S phase upon DONSON depletion. HeLa cells were treated with siControl or siDONSON for 48 h, and soluble proteins were pre-extracted by CSK buffer on ice for 10 s before fixation. i Quantification of the normalized signal intensity of chromatin-bound LRR1 in S phase observed in (h). n = 50 from three independent experiments. j Inducible expression of RNAi-resistant mNG-DONSON or the indicated deletion mutant upon DONSON depletion. k Quantification of the normalized signal intensity of phosphorylated Plk1 (T210) at each centriole with the indicated treatment. n = 50 from three independent experiments. All scale bars, 5 μm. Values are mean percentages ± s.d. Mann–Whitney U test was used in (i) and the Kruskal–Wallis test was used in (k) to obtain the P-value. Source data are provided as a Source Data file.

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