Figure 6.

(See previous page). Cdt2-mediated XPG degradation facilitates the recruitment of DNA Pol δ to UV-damaged sites and subsequent gap-filling DNA synthesis. (A and B) Cdt2 is not required for the removal of UV-induced DNA lesions. HeLa cells were transfected with either control siRNA or siCdt2 for 48 h. Cells were UV irradiated at 10 J/m², and further cultured for the indicated time periods. Genomic DNA was isolated and the same amount of DNA was loaded for ISB with anti-CPD or anti-6-4PP antibody. Single strand DNA (ssDNA) was detected to serve as a loading control. (A) The intensity of each band was scanned and plotted. (B) (C and D) Cdt2 is required for the gap-filling DNA synthesis upon UV irradiation. HeLa cells growing on coverslips were transfected with either control or Cdt2 siRNA for 48 h, UV irradiated at 100 J/m² through a 5 μm isopore filter and maintained in medium containing BrdU for 2 and 4 h. The cells were fixed and double immunostained with anti-CPD (green) and anti-BrdU (red) antibodies. (C) The total numbers of CPD and BrdU foci in non-S phase cells were counted from at least 5 separate fields, the ratios of BrdU to CPD foci were calculated and plotted. (D) Yellow arrow: S phase cells. Bar: SD, *: P < 0.01 compared with their corresponding siCtrl. (E and F) Cdt2 is required for the recruitment of DNA Pol δ to UV damaged sites. siCtrl and siCdt2 transfected HeLa cells were UV irradiated at 100 J/m² through a 5 μm isopore filter and maintained in medium for 1 h. The cells were fixed and double immunostained with anti-XPB (green) and anti-p125 (red) antibodies. (E) The total numbers of XPB and p125 foci were counted and the ratios of p125 to XPB foci were calculated and plotted. (F) n = 5, Bar: SD, *: P < 0.01 compared with siCtrl.