Figure 1: PARP3 promotes chromosomal SSBR and is stimulated by SSBs with canonical termini.

(a) Wild-type (WT) DT40 cells, PARP3^−/− (PARP3 KO) DT40 cells, and PARP3^−/− DT40 cells stably transfected with either empty vector (vector) or vector encoding human recombinant PARP3 (hPARP3) were treated with the indicated doses of γ-rays and survival calculated in clonogenic assays. Data are the mean (±s.e.m.) of three independent experiments. Where not visible, error bars are smaller than the symbols. (b) WT, PARP1^−/−, or PARP3^−/− DT40 cells were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified (tail moment) by alkaline comet assays. Data are the average tail moment of >50 cells per sample and are the mean of three independent experiments (±s.e.m.). (c) WT, PARP3^−/−, or derivatives of PARP3^−/− DT40 cells complemented with empty vector or hPARP3 were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified as above. (d) WT, KU70^−/−, or XRCC3^−/− DT40 cells were treated on ice with γ-rays (20 Gy) and incubated for the indicated times to allow repair. DNA strand breaks were quantified as above. ANOVA was employed to compare mutant DT40 for significant differences with WT (**P<0.01. ‘ns’; not significant). Data are the mean (±s.e.m.) of three independent experiments. (e) hPARP1 and/or hPARP3 (50 nM) was incubated with 12.5 μM biotin-NAD⁺ and 200 ng uncut or nicked plasmid (nicked with Nt.BsmA1; nick concentration of 32 nM) that was pretreated or not as indicated with CIP to dephosphorylate 5′-termini. Reaction products were separated by SDS–PAGE and blotted with streptavidin-HRP. (right) Aliquots of uncut, nicked, and linear plasmid were analysed by agarose gel electrophoresis and staining with ethidium bromide. ANOVA, analysis of variance; CIP, calf intestinal phosphatise; HRP, horseradish peroxidase