Fig. 2: Repair of DNA double-strand breaks in COMMD4-depleted cells and interaction with hSSB1.
From: COMMD4 functions with the histone H2A-H2B dimer for the timely repair of DNA double-strand breaks
a Comet assay showing the relative comet tail moment in COMMD4-deficient and control cells at 0 and 0.5 h post treatment with 6 Gy IR. b Plot of the relative NHEJ events in control, cells treated with DNA-PK inhibitor, COMMD4-depleted cells, COMMD4-depleted cells transiently overexpressing COMMD4-FLAG and cells stably expressing COMMD4-FLAG, 48 h post-transfection with HindIII linearised EGFP-N3 plasmid. c Plot of I-Sce1 induced HR in control, hSSB1-depleted, COMMD4-deficient cells and COMMD4-deficient cells overexpressing COMMD4-FLAG transiently and stably. d Plot of the relative NHEJ and HR events, respectively, in control, DNA-PK inhibitor, hSSB1 siRNA and COMMD4-depleted cells using a quantitative reporter assay that measures NHEJ versus HR in the same cells through the repair of two inverted I-Sce1 cuts. e A direct interaction between COMMD4 and WT hSSB1, T117E and S134E hSSB1. COMMD4 was used to pull-out the protein complexes. IgG is the negative control and COMMD4 shows the loading. f ChIP-qPCR assay with primers at 94–378 and 300–643 bp from the break-site in DRGFP cells transfected with COMMD4-FLAG, ± I-Sce1. The enrichment of COMMD4 after induction of a DSB was compared to the IgG control and - I-Sce1 sample. Significant recruitment was only observed at 300–643 bp. The schematic of the assay and the primer pairs used is shown below. *p < 0.05, **p < 0.005, ns non-significant where p > 0.05. Error bars represent mean ± SD from three independent experiments. g Immunofluorescence showing localisation of FLAG-COMMD4 and RNF20 at a DSB induced by the I-SceI restriction enzyme in MCF7 DRGFP cells. Scale bar denotes 5 μm.
