Article Figures & Data
Figures
- Figure S1. Characteristics of DSCC1-KO cells.
(A) Alignment of sequences of WT and DSCC1-KO cells at the crDSCC1 target site. The red dotted line indicates the PAM site, and the black dotted line indicates the Cas9 cut site. (B) Cell cycle analysis of WT and DSCC1-KO cells using flow cytometry. Mitotic cells (M) are identified by phosphorylation of histone H3 at serine 10. Actively replicating cells (S) are identified by EdU incorporation, and G1 and G2 are separated by DNA content as stained by DAPI. (C) Proliferation of DSCC1-KO cells relative to WT cells was assessed by CTB assay after 5 d of proliferation. Dots indicate the mean calculated from three technical replicates from three independent experiments.
- Figure 1. Human DSCC1-RFC has roles in sister chromatid cohesion, DNA replication, and DNA repair.
(A) SCC defects of indicated cell lines. At least 50 metaphases were scored per condition; three independent experiments are shown as separate bars. (B) DNA replication fork speed of indicated cell lines was assessed with a DNA fiber assay. At least 65 fibers were scored per experiment per condition in two independent experiments. Indicated P-value is calculated with an unpaired t test. Scale bar represents 5 µm. (C) Cells were assessed for γH2AX foci using immunofluorescence. At least 47 cells were scored per experiment per condition in three independent experiments, and the percentage of cells with more than five foci is shown. The P-value is calculated with an unpaired t test. Scale bar represents 5 µm. (D) WT and DSCC1-KO cells were treated with indicated drugs and proliferation relative to untreated cells was assessed after 5 d by a CTB assay. Three technical replicates from a representative of two independent experiments are shown.
Source data are available for this figure.
Source Data for Figure 1[LSA-2022-01596_SdataF1.xlsx]
- Figure 2. Genome-wide CRISPR screen reveals multiple synthetic lethal interactions of DSCC1-KO cells.
(A) Network of hits synthetically sick/lethal with DSCC1-KO as determined by DrugZ analysis with an FDR < 0.1. Edges indicate physical protein–protein interactions as annotated in Genemania.org. (B) Gene Ontology enrichment of <0.1 FDR hits synthetically sick/lethal with DSCC1-KO extracted from String-db. (C) WT and DSCC1-KO cells were transfected with indicated crRNAs and treated with doxycycline to induce Cas9 expression. After 7 d, cell viability was assessed by CTB assay and normalized for cells depleted of the nonessential olfactory receptor OR10A7. POLR2L was included as a common essential control. Dots indicate means calculated from three technical replicates of three independent experiments. (D) Clonogenic survival of WT and DSCC1-KO cells 11 d after crRNA transfection and Cas9 induction. Figure shows a representative of two independent experiments.
Source data are available for this figure.
Source Data for Figure 2[LSA-2022-01596_SdataF2.xlsx]
- Figure S2. Genetic relationship between POLE3 and DSCC1.
(A) Pearson correlation of co-dependency score between cell lines of the top four genes correlating with DSCC1 extracted from depmap.org. (B) Alignment of sequences of WT, POLE3-KO1, and POLE3-KO2 cells at the crPOLE3 target site. The red dotted line indicates the PAM site, and the black dotted line indicates the Cas9 cut site. Note that alignment at the crPOLE3 site for POLE3-KO1 is completely lost because of a 125 bp deletion. (C) CRISPR editing efficiency at the crPOLE3 target site resulting in KO indels (out of frame or >26 bp deletion) or in frame indels. (D) SCC defects of indicated cell lines. At least 50 metaphases were scored per condition. (E) SCC defects of indicated cell lines, after transfection with indicated crRNAs. At least 50 metaphases were scored per condition, and two independent experiments are shown as separate bars. (F) Percentage of mitotic cells assessed by FACS analysis of histone H3 phosphorylated at serine 10.
- Figure 3. POLE3 and DSCC1 contribute to DNA replication fork progression in an additive manner.
(A) Relative viability of WT, DSCC1-KO, and two POLE3-KO clones treated with ATR inhibitor (ATRi, AZD6738) or topoisomerase I inhibitor camptothecin assessed by CTB 5 d after addition of drug. Dots represent three technical replicates from a representative of two independent experiments. (B) Replication fork speed of POLE3-KOs assessed by a DNA fiber assay. At least 80 fibers were scored per condition. P-values were determined by an ordinary one-way ANOVA. (C) Cells were assessed for γH2AX foci using immunofluorescence. At least 42 cells were scored per experiment per condition in three independent experiments, and the percentage of cells with more than five foci is shown. Scale bar represents 5 µm. (D) WT and DSCC1-KO cells were transfected with indicated crRNAs and treated with doxycycline to induce Cas9 expression. After 5 d, DNA replication fork speed was assessed by a fiber assay. At least 50 fibers were scored per experiment per condition in two independent experiments. Indicated P-values are determined by an ordinary one-way ANOVA. (E) Flow cytometry analysis of WT and DSCC1-KO cells 5 d after transfection with indicated crRNA:tracrRNAs. Actively replicating cells (S) are identified by EdU incorporation and G1 and G2 by DNA content stained by DAPI.
Source data are available for this figure.
Source Data for Figure 3[LSA-2022-01596_SdataF3.xlsx]
- Figure 4. MMS22L-TONSL heterodimer contributes to sister chromatid cohesion in parallel with DSCC1-RFC.
(A) Cohesion analysis of indicated cell lines 5 d after transfection of indicated crRNA:tracrRNA complexes with induction of Cas9. Each bar represents an analysis from three independent experiments. At least 50 metaphases were analyzed per condition. (B) Cells were transfected with indicated crRNAs and treated with doxycycline to induce Cas9. After 2 d, cells were treated with 2 mM thymidine for 24 h and analyzed by flow cytometry. (B, C) Western blot of chromatin-bound and soluble protein fractions from cells treated as in (B). (D) Relative viability assessed by CTB 7 d after transfection of indicated crRNA:tracrRNA complexes with induction of Cas9. Data points represent means calculated from three technical replicates from three independent experiments. Indicated P-values were calculated by an ordinary one-way ANOVA. (E) Clonogenic assay 11 d after transfection of indicated crRNA:tracrRNA complexes with induction of Cas9. (F) Proposed model of DSCC1-RFC and MMS22L-TONSL functions in SCC establishment pathways. DSCC1-RFC facilitates the de novo cohesin loading pathway, whereas DDX11 and MMS22L-TONSL contribute to the cohesin conversion pathway. ESCO2 can be recruited to chromatin by PCNA, which may be loaded by DSCC1-RFC. MMS22L can also recruit ESCO2 to the replication fork, possibly by binding to newly replicated chromatin. The fact that MMS22L-TONSL and DSCC1-RFC contribute to different SCC pathways may suggest that different ESCO2 recruitment mechanisms preferentially contribute to different SCC pathways.
Source data are available for this figure.
Source Data for Figure 4[LSA-2022-01596_SdataF4.1.xlsx]
Source Data for Figure 4[LSA-2022-01596_SdataF4.2.pdf]
Supplementary Materials
Table S1 CRISPR screen count data.
Table S2 Supplemental materials.
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