Fig 2: MCPH1 isoforms are phosphorylated during mitosis. A) U2OS cells were treated with 100 ng/ml nocodazole for the indicated time points before Western blot analysis. Asynchronous cells (Asy) were also analyzed. B) U2OS cells were synchronized in prometaphase by treatment with 100 ng/ml nocodazole (20 h). RO3306 (9 µM) was then added to the culture medium for the indicated time points before Western blot analysis. C) U2OS cells were treated with nocodazole (100 ng/ml) for 20 h before Western blot analysis. In the right-hand panel, nocodazole-treated cells were lysed in native lysis buffer before addition (or not) of ?-phosphatase for 20 min at 30°C. Samples were then analyzed by using Western blotting. D) Samples from B were reanalyzed by Western blotting to allow comparison between 2 different MCPH1 antibodies (Cell Signaling Technology/CST vs. Proteintech). Aby, antibody.

Fig 3: Mcph1-?e8 cells exhibit PCC and defective DDR. (A) Representative images of PCC in Mcph1-?e8 MEFs. Primary MEF cells were stained with a pS10-H3 antibody (red) and counterstained with DAPI (blue). (B) Quantification of the percentages of PCC cells (prophase cells lacking the pS10-H3 signal) in control and Mcph1-?e8 primary MEF cells. More than 250 prophase cells were scored in each group of the indicated genotype. (C) Western blot analysis of p-Chk1 and ?H2AX in control and Mcph1-?e8 MEFs with or without HU treatment. ß-actin was used as a loading control. (D) The quantification of the indicated protein intensities from panel (C). Unpaired Student’s t-test was used for statistical analysis. ***, p < 0.001.

Fig 4: MCPH1 prevents both RAD51- and DMC1-ssDNA filament disassembly. (A) Schematic of our tethered particle motion disassembly experiments. The surface-bound DNA with bead was pre-incubated with RAD51-ATP or DMC1-ATP to form nucleoprotein filaments. Free recombinases were washed away and then tag-free MCPH1 was added. Disassembly of recombinase-ssDNA filaments was monitored in real-time by characterizing the change in time-lapsed Brownian motion (BM) of the DNA tethers. (B, C) Representative BM time traces of RAD51–ssDNA filament disassembly in the absence of MCPH1 (B) and in the presence of 25 nM MCPH1 (C). (D) Survival probability histograms of RAD51–ssDNA filament lifetime at different MCPH1 concentrations. AMP-PNP was included to replace ATP for RAD51 as a positive control. Fitted curves are based on the Kaplan-Meier estimator. (E) Bar graph of RAD51–ssDNA filament lifetime obtained from D. (F, G) Representative BM time traces of DMC1-ssDNA filament disassembly in the absence of MCPH1 (F) and in the presence of 25 nM MCPH1 (G). (H) Survival probability histograms of DMC1-ssDNA filament lifetime under different conditions. (I) Bar graph of DMC1-ssDNA filament lifetime compiled from (H). Data include at least 32 DNA tethers from at least three independent experiments. Error bars represent the standard deviation (±SD). Symbol meaning: *P = 0.05; **P = 0.01.

Fig 5: Expression, purification, and identification of MCPH1 protein. (A) The expression plasmid of MCPH1. (B) The MCPH1 expression plasmid was transfected and expressed in human Expi293F cells. Whole cell extract was analyzed in a 10% SDS-denaturing polyacrylamide gel and stained with Coomassie Blue (stain) or western blot with anti-MCPH1 antibody (blot). (C) Schematic of our purification protocol. The Expi293F cell extract was fractionated through amylose, Ni+-NTA, phenyl HP and heparin columns. The MBP tag was removed using GST-tagged PreScission protease and then filtered through a GST column to remove the protease. (D) Purified MCPH1 protein (1 µg), as analyzed in a 12% SDS-denaturing polyacrylamide gel with Coomassie Blue staining. (E) Results from ESI-MS/MS analysis of purified MCPH1. Identified fragments are shaded in gray.