Figure 8

Deletion of Polθ-helicase domain overexpression increases the recruitment of MCM to DNA and impairs DNA replication. (A) Schematic representation of deletion of the helicase domain using CRISPR-CAS9 technology. We generated a double-stranded break within the helicase domain and provided a 120 bp fragment containing the 5′ and 3′ regions of the helicase domain as a donor. (B) Proteic extracts of wild-type cells and cells from the lineage in which the helicase domain was deleted (Δ Helicase domain) were subjected to western blotting using anti-Tc polθ-helicase antibody. (C) Wild-type and Δ Helicase domain cells were submitted to cell fractionation, during which soluble proteins were discarded, and DNA was treated with DNase to release DNA-bound proteins. DNA-bound proteins were subjected to western blotting using anti-MCM7 and anti-histone H3 as the loading control. (D) The bands present in (C) were quantified, and the values are expressed as the median and standard deviation of three independent experiments. (E) The graph shows the percentage of cells that incorporated EdU. Values are expressed as the median and standard deviation of three independent experiments. One hundred cells were analyzed in each replicate.