Fig. 7: DCAF15 loss disrupts DNA replication fork integrity.

a Workflow of DNA fiber analysis for quantification of replication fork restart efficiency after 1 mM hydroxyurea (HU)-induced replication fork stalling. Cells cultured in the presence of CldU for 20 min prior to addition of HU. After 60 min, cells washed with PBS prior to adding Idu for 40 min. Representative images of DNA fibers shown for Cas9+ HEL cells infected with ROSA26- or DCAF15-targeting sgRNAs. b Quantification of (a) shows IdU/CldU ratios (replication fork restart efficiency) in individual experimental conditions (sgROSA26: n = 154 fibers analyzed, sgDCAF15#2: n = 278 fibers analyzed). Median IdU/CldU ratios labeled and indicated by horizontal blue (sgROSA26) and red (sgDCAF15#2) bars. Two-tailed Mann–Whitney test, ****p-value < 0.0001. c Cas9 + MV4-11 TP53-WT and TP53-knockout (sgTP53#6) cells infected with ROSA26- or DCAF15-targeting sgRNAs harvested 4 days post lentiviral sgRNA infection. Cytoplasmic (Soluble) and nuclear (Chromatin) protein fractions analyzed by Western blot for indicated proteins. Immunoblots representative of three independent experiments. d Competition-based proliferation assay performed in Cas9 + MV4-11 cells infected with ROSA26- or DCAF15-targeting sgRNAs were grown in presence of 1 nM camptothecin (CPT). sgRNA+ populations monitored over time for mCherry expression by flow cytometry. Plotted values are relative sgRNA+ proportions growing with CPT normalized to matched sgRNA+ growing with DMSO. Error bars mean ± SD, n = 3 biologically independent replicates. Two-way ANOVA (with Bonferroni’s multiple comparisons test), ****padj < 0.0001, ns not-significant. e Same as in (d), except cells grown in the presence of olaparib (Ola) for 7 days. Relative sgRNA+ proportion quantification is shown for cells growing with olaparib normalized to matched sgRNA+ proportion growing with DMSO. Error bars mean ± SD, n = 3 biologically independent replicates. Two-way ANOVA (with Bonferroni’s multiple comparisons test), ****padj < 0.0001, ns not-significant. f HDAC8-mediated cohesin deacetylation enables CRL4-DCAF15 recruitment. DCAF15-mediated destabilization of cohesin-bound PDS5A and CDCA5 enables timely ESCO1/2-mediated cohesin re-acetylation, vital for accurate halting of DNA loop extrusion, efficient DNA replication fork progression, and sustained AML proliferation (Left). In the absence of DCAF15, HDAC8-de-acetylated cohesin is inappropriately hyper-loaded with PDS5A and CDCA5, which precludes ESCO1/2-mediated cohesin re-acetylation and leads to uncontrolled DNA loop extrusion, defective DNA replication fork progression, and activation of apoptosis (Right). Source data are provided as a Source Data file.