Journal: bioRxiv
Article Title: MCM10 and SLD-2/RECQL4 jointly activate the CMG helicase during metazoan DNA replication initiation
doi: 10.64898/2026.04.01.715773
Figure Lengend Snippet: ( A - C ) Control and BromoTag-Mcm10 dTAG-Recql4 mouse ES cells were incubated for eight hours in the presence or absence of the PROTACs AGB1 and dTAG13, or in the presence of 2 mM hydroxyurea, before treatment for 20 minutes with EdU. Cells were analysed by high-content screening microscopy as described above for . For panels (B) and (C), EdU-negative cells (see Methods) are indicated in black. ( D ) Samples for the above high-content screening experiment were also analysed by immuno-staining with phospho-specific antibodies corresponding to Serine 4 (S4) and Serine 8 (S8) of RPA32, which is phosphorylated in response to activation of the DNA replication checkpoint pathway. ( E ) Samples from the same experiment were used to prepare cell extracts that were analysed by immunoblotting for the indicated proteins. ‘P-CHK1 (S345)’ corresponds to phosphorylation of Serine 345 of CHK1, whereas ‘P-RPA32 (S33)’ corresponds to phosphorylation of Serine 33 of RPA32, both of which are mediated by the ATR kinase in response to checkpoint activation. ( F ) Mouse ES cells of the genotype GFP-Psf1 (Control) or BromoTag-Mcm10 dTAG-Recql4 GFP-Psf1 were incubated for four hours in the presence or absence of the PROTACs AGB1 and dTAG13. Live cells were then stained with the DNA-binding dye SPY650-DNA (see Methods). Arrows indicate the accumulation of GFP-PSF1 on patches of constitutive heterochromatin. The scale bar corresponds to 10 µm.
Article Snippet: For the experiment in , live mouse ES cells were stained with a 4000 fold dilution of SPY650-DNA (SC501, Spirochrome), before imaging by spinning-disk confocal microscopy as described above for C. elegans .
Techniques: Control, Incubation, High Content Screening, Microscopy, Immunostaining, Activation Assay, Western Blot, Phospho-proteomics, Staining, Binding Assay
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