PARP1 and DSB-interacting proteins co-localize at DNA damage sites. SK-N-SH cells were fixed 30 min after laser microirradiation. a, endogenous PARP1 was colocalized with the DSB marker 53BP1. Inset, image of a single microirradiated cell. Nuclei were stained with DAPI. b, induction of a single DSB in SK-N-SH cells was performed through transfection of the I-SceI expression vector pCBASce, and immunofluorescence was conducted with antibodies against PAR and MRE11. Inset, PAR synthesis and MRE11 assembly at a unique DSB. Bar, 3 μm. c, the presence of endogenous PARP1 on a unique DSB was verified by ChIP analysis followed by real time PCR quantification at 675-1044, 3014-3207, and 4545-4727 nucleotides from the break (red, yellow, and blue bars, respectively). The graph represents the relative -fold increase of the PARP1 protein compared with an IgG control. The PCRs were performed in triplicate. d, schematic representation of the position of primers used for real time PCR quantification relative to the unique DSB created by I-SceI in vivo.
