Fig 2: Phosphorylation of CBX3 serine-95 by CK2 kinase augments CDH1-mediated CBX3 degradation and RPA2 dynamics at stalled replication forks.(A) WB analysis of Flag-CBX3 proteins in PC-3 cells pulled down by GST or GST-CDH1 recombinant proteins. (B) Diagram showing phosphorylation motifs from the D-box in CBX3. (C) WB analysis of WCL derived from PC-3 control and CK2 knockdown cells. (D and E) PC-3 control or CK2 knockdown cells were treated with CHX (50 μg/μl) for WB (D). Protein bands were quantified as in (E). (F) MS analysis reveals phosphorylation of CBX3 serine-95. (G) WB analysis of WCL and anti-Flag IPs derived from PC-3 cells transfected with indicated plasmids. (H) WB analysis of WCL and anti-Flag IPs derived from PC-3 cells transfected with indicated plasmids. (I and J) PC-3 cells transfected with indicated plasmids were treated with CHX (50 μg/μl) for WB (I). Protein bands were quantified as in (J). (K to M) U2OS control or CBX3 knockdown cells were reconstituted with WT or V22M mutant CBX3 and subjected to IFC after HU treatment. Representative images were shown in (K). Scale bar, 10 μm. Quantitative data of RFWD3 foci were shown in (L) and RPA2 foci were shown in (M). Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student’s t test, ***P < 0.001.

Fig 3: The stability of SUMOylation-deficient MRE11 is improved by fusion with a poly-SUMO2 chain.a Schematic of the 4KR-MRE11 protein with a 3×SUMO2 chain fused to the C-terminus. Fusion protein expression was detected by immunoblotting. b The ubiquitylation of 4KR was inhibited by fusion with the 3×SUMO2 chain. c SUMOylation-deficient MRE11 fused with poly-SUMO2 facilitated RPA2 phosphorylation and ATR-CHK1 pathway activation after DNA damage. shMRE11 HeLa cells reconstituted with SFB-WT, SFB-4KR, and SFB-4KR-poly-SUMO2 as indicated were treated with 1 μM CPT for 1 h or not. d Cell viability was determined by CCK-8 assay after treatment with 0.5 μM CPT or 1 μM cisplatin for the indicated times (means ± SEM, n = 3 independent experiments).

Fig 4: CBX3 antagonizes RFWD3-mediated RPA2 polyubiquitination and promotes RPA2 retention at stalled replication forks.(A and B) U2OS cells infected with lentivirus expressing control or shCBX3 were subjected to IFC after HU treatment. Representative images were shown in (A) and quantitative data are shown in (B). Scale bar, 10 μm. Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student's t test, ***P < 0.001. (C) Replication fork-bound proteins were isolated by the iPOND assay with control and CBX3 knockdown U2OS cells. (D) WB analysis of WCL and anti-His IPs derived from PC-3 cells transfected with indicated plasmids followed by HU treatment. (E) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment. (F) WB analysis of WCL and anti-His IPs derived from PC-3 cells transfected with indicated plasmids followed by HU treatment. Both poly-ubiquitinated and primary-ubiquitinated RPA2 (asterisk) were observed. (G) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment. Both poly-ubiquitinated and primary-ubiquitinated RPA2 (asterisk) were observed. (H and I) U2OS control or CBX3 knockdown cells were reconstituted with WT or V22M mutant CBX3 and subjected to IFC after HU treatment. Representative images were shown in (H) and quantitative data were shown in (I). Scale bar, 10 μm. Data were shown as the means ± SD from three biological replicates, two-tailed unpaired Student's t test, ***P < 0.001. (J) WB analysis of WCL and chromatin binding proteins derived from PC-3 control and CBX3 knockdown cells transfected with indicated plasmids followed by HU treatment.

Fig 5: CBX3 is recruited to stalled replication forks in an RPA2-dependent manner.(A) FLAG-tagged CBX3 was immunoprecipitated from 293T cells and subjected to MS analysis to identify CBX3 interacting proteins. (B) The list of top hits proteins from CBX3 MS. (C) Western blot (WB) analysis of whole-cell lysates (WCL) and anti-CBX3 immunoprecipitations (IPs) derived from PC-3 cells. (D) WB analysis of WCL and anti-RPA2 IPs derived from PC-3 cells. (E) Colocalization of CBX3 with RPA2 foci after noted drug treatment or transfected with indicated small interfering RNA (siRNA) in U2OS cells. Scale bar, 10 μm. (F) Quantitation of average CBX3 foci number in each cell. Data were presented as means ± SD of more than 200 cells from three biological replicates. n.s., not significant. Two-tailed unpaired Student’s t test, ***P < 0.001. (G) Replication fork-bound proteins were isolated by the iPOND assay with control and RPA2 knockdown U2OS cells. (H) Schematic diagram depicting a set of GST-RPA2 recombinant protein constructs. (I) WB analysis of Flag-CBX3 proteins in U2OS cells pulled down by GST or GST-RPA2 recombinant proteins. (J) Schematic diagram depicting a set of GST-CBX3 recombinant protein constructs. (K) WB of His-RPA2 proteins in U2OS cells pulled down by GST or GST-CBX3 recombinant proteins. (L and M) U2OS cells transfected with Flag-CBX3 proteins were subjected to immunofluorescence staining (IFC) after HU treatment. Representative images were shown in (L) and quantitative data are shown in (M). Scale bar, 10 μm. Data were shown as the mean ± SD from three biological replicates, two-tailed unpaired Student’s t test, ***P < 0.001. IgG, immunoglobulin.