Fig. 6: Loss of RECQL4 diminishes the DSBR pathways NHEJ and alt-NHEJ.

a, The effect of PARP1 and its activity on the chromatin binding of RECQL4 after DNA damage. Chromatin fractions were prepared from U2OS WT/PARP1 KO cells before and after 10 Gy of IR DNA damage with or without 5 µM olaparib treatment. Western blot analysis was performed using an anti-RECQL4 antibody. Anti-H3 served as a chromatin marker and loading control. The graph shows the quantitative representation of chromatin-bound RECQL4. A Student’s t-test (two-sided) was performed to assess statistical significance (*P < 0.05, **P < 0.01 and ***P < 0.001). b, ChIP analysis of RECQL4 occupancy at AsiSi-induced DSBs. ChIP analysis was performed in AsiSi–ER–U2OS cells before (−) and after (+) 4 h of 4-OHT treatment and with or without PARP1 inhibition (Ola) or knockdown (siPARP) using rabbit IgG or anti-RECQL4 antibodies. Enrichment was assessed via real-time qPCR amplification using primers proximal and distal to the AsiSi-induced DSB site on a specific chromosome. The error bars represent the s.e.m. A two-way ANOVA was performed to assess significant differences (*P < 0.05, **P < 0.01 and ***P < 0.001). c, d, Schematic images of the results of the cellular GFP reporter cassette DNA repair assays, with the effects of RECQL4, BLM and PARP1 inhibitors on the efficiency of repairing I-SceI-mediated DSBs through the c-NHEJ (c) and alt-NHEJ (d) pathways. RECQL4 inhibits NHEJ with or without a PARP1 inhibitor (olaparib) 24 h post-siRNA transfection (c). EJ5 cells were cotransfected with plasmids expressing I-SceI and DsRed constructs, and the relative NHEJ efficiency was measured. RECQL4 inhibits alt-NHEJ in PARP1 inhibitor (olaparib)-treated cells (d). Plasmid-transfected cells were treated with olaparib for 4 days, after which NHEJ and alt-NHEJ efficiency were measured. The error bars represent the s.e.m. of three independent experiments. The repair efficiency of each repair pathway is reported relative to the siControl condition, which is set arbitrarily to 1.0. All experiments were repeated at least three times, and the error bars represent the s.e.m. A two-way ANOVA was performed to assess significant differences (*P < 0.05, **P < 0.01 and ***P < 0.001). e, A schematic outline of the MMEJ in vitro assay (left). Mean number ± s.d. of colonies obtained from the in vitro MMEJ assay using nuclear lysates from U2OS cells expressing Flag–RECQL4 alone or with V5-PARP1 in the presence or absence of GFP–PARG after 10 Gy IR from three independent experiments; ***P < 0.001 and ****P < 0.0001 using one-way ANOVA (right top). Representative images of colonies harboring the repaired pBABE-hygro-MMEJ plasmid (right bottom).