Figure 6.

LIG3 knockdown and the recruitment of XRCC1 and PNKP to sites of DNA damage. Laser micro-irradiation was performed on HeLa cells expressing reduced levels of LIG3 (see Supplementary Figure S5) and (A) XRCC1-mRFP or (B) PNKP-mRFP. Reduced background levels of LIG3 lead to decreased overall recruitment of XRCC1 and PNKP to sites of DNA damage. (C) Simultaneous inhibition of PARP1 (using AG14361) and knockdown of LIG3 showed an additive effect on the reduction of the amount of PNKP recruited to sites of DNA damage. For recruitment curves, error bars represent SEM from three independent experiments for a total of 36 individual cells. Note that the mRFP photobleaches during laser micro-irradiation resulting in an initial loss of fluorescence at the damage sites.