Fig. 6
From: PARP2 mediates branched poly ADP-ribosylation in response to DNA damage
PARP2-dependent branched PAR chain is important for histone H3 removal in response to DNA damage. a Doxycycline (DOX)-inducible Cas9 (iCas9) was expressed in the U2OS or U2OS PARP2-null cells. The expression of iCas9 was examined by western blot using anti-FLAG antibody (monoclonal antibody, Sigma F1804, the inset). A gRNA facilitates a single DSB as the AAVS1 locus. P1 represents a pair of primers flanking both sides of the DSB for the analysis of DSB repair. P2 is a pair of primers specific to a region 2 kb upstream to the DSB and serves as a negative control of DSB repair. P3 and P4 are two sets of primers located at 0.3 kb upstream and downstream respectively from the DSB. These two primers were used for the detection of histone H3 removal in response to DNA damage. b γH2AX (polyclonal antibody, Abcam ab11174) was examined as a surrogate maker of the solo DSB at AAVS1 locus using immunofluorescence analysis. c Histone H3 removal is severely compromised in the absence of PARP2. Histone H3 removal was examined by ChIP assays with antibody (polyclonal antibody, Millipore 06-755) using P3 and P4 primers. d Depletion of APLF also suppressed histone H3 removal at the DSB. Depletion of APLF was verified by western blot using anti-APLF antibody (polyclonal antibody, Thermo Fisher Scientific PA5-39776, left panel). e DSB repair was monitored by q-PCR using P1 and P2. f Loss of either PARP2 or APLF significantly suppresses DSB repair. DSB repair in cells lacking PARP2 or APLF was examined by q-PCR. Scale bar represents 5 μm
