Fig. 5

Methylation of PCNA at lysine 110 affects the binding of DNA polymerase δ to PCNA. a A 3 h EdU pulse was applied in EZH2-knockdown hDPCs at 21 h after release from serum deprivation. Scale bars represent 100 μm. b Formaldehyde protein crosslinking assay indicates the presence of the trimer form of PCNA in EZH2-depleted 293T cells. The left panel illustrates the location of lysine 110 on the “head” β-sheet of the interface between the homo-monomers. c Crosslink assay shows the level of the trimer form of PCNA in 293T cells overexpressing EZH2∆SET and EZH2∆PIP. d IP demonstrates the decreased interaction between PCNA and POLδ when knocking down EZH2 or overexpressing EZH2∆PIP and EZH2∆SET in 293T cells. e Schematic representation of full-length PCNA with a point mutation at lysine 110 (K110). A Myc tag was added on the C-terminus. The crosslink assay shows the level of trimer PCNA upon lysine 110 mutation (PCNA∆). IP demonstrates the binding of POLδ to PCNA when PCNA∆ is overexpressed in 293T cells. f DNA fibre assay demonstrates DNA synthesis in cells expressing the lysine 110 mutant (PCNA∆). Origins are indicated by arrows. Scatter plot and bar chart show the DNA length (***P < 0.001). IgGs were used as a control antibody for IPs. Antibodies used for IP and western blot are labelled as IP and IB, respectively