Figure 3: The PARP3 DNA-binding interface is required for PARP3 stimulation and accumulation at chromosome DNA damage.

(a) Wild-type or the indicated mutant full-length cPARP3 (300 nM) was incubated for 20 min at room temp with biotin-NAD⁺ (12.5 μM) and 200 nM of oligonucleotide duplex harbouring either a 5′-phosphorylated nick or 5′-phosphorylated DSB with 3′-overhang. Reaction products were separated by SDS–PAGE, blotted, and detected with streptavidin-HRP. Autoribosylated cPARP3 was quantified and plotted relative to that generated in reactions containing nicked duplex and wild type cPARP3. Data are the mean (±s.e.m.) from three independent experiments. (b) Time-course of wild-type or mutant cPARP3 incubated from 0 to 30 min in the same conditions as above. (c) Recombinant wild-type or mutant cPARP3 (0–0.8 μM) was incubated with a 3′-fluorescein isothiocyanate (FITC)-labeled oligonucleotide duplex harbouring a 5′-phosphorylated nick (100 nM), and protein-DNA complexes detected by EMSA. (d) Recruitment of wild-type and mutant human PARP3-GFP to sites of UVA-laser DNA damage in human U2-OS cells. (left) Representative images of WT and mutant PARP3-GFP before treatment (Unt) and 1 min after laser damage. (top right) Quantification of GFP accumulation at sites of laser damage (% increase over initial level). Data are the mean (±s.e.m.) of 25 or more cells per sample. The hPARP3 WGR mutations were Y83A, W101L and R103N and H384A/E514A in the catalytic domain (denoted ‘CM’). (e, top) PARP3^−/− DT40 cells stably transfected with either empty vector (vector) or vector encoding wild-type hPARP3 (WT) or the mutant derivatives Y83A, W101L and R103N 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. The inset is a western blot showing the expression level of wild type and mutant hPARP3 in PARP3^−/− DT40 cells. (bottom) The above DT40 cell lines were treated with the indicated doses of γ-rays and survival quantified in clonogenic assays. Data are the mean (±s.e.m.) of three independent experiments. Where not visible, error bars are smaller than the symbols. HRP, horseradish peroxidase.