Fig. 2.
Defective repair of UV photo lesions in cells lacking INO80 and transcriptional impact of INO80 depletion. (A) Slot blotting of CPD (Left) in genomic DNA isolated from INO80 WT and INO80 mutant cells (4H12 and 2D15) irradiated with 8 J/m2 of UV (254 nm) and harvested at 0, 6, and 24 h. Quantifications of the relative CPD levels were arrived at by normalizing the chemiluminescent signal against the total DNA signal obtained by Southern hybridization with a total genomic DNA probe (Right). (B) Slot blotting of 6-4PP (Left) in genomic DNA isolated from INO80 WT and INO80 mutant cells (4H12 and 2D15) irradiated with 8 J/m2 of UV (254 nm) and harvested at 0, 1, 3, and 6 h. Quantifications of the relative 6-4PP levels were similarly performed as in A. (C) Immunoblotting of NER proteins in INO80+/+ and INO80−/− cells. (D) In vitro NER activity of INO80 and ARP5 mutants. Nuclear extracts were prepared from wild-type (INO80+/+), heterozygous (P1G8, INO80Flox/+), and two conditional INO80Flox/− cells (4H12 and 2D15) treated (+) and untreated (−) with AdCre and used in the NER synthesis assay. (E) Repair synthesis assay with nuclear extracts prepared from WT (ARP5+/+) and two conditional ARP5Flox/− mutants (7B1 and 5E9) treated (+) or untreated (−) with AdCre. (Upper) DNA substrates from each sample were recovered after the repair synthesis assay and resolved on an agarose gel. (Lower) Autoradiography generated by PhosphorImager (Molecular Dynamics). Relative NER activity of each sample (Bottom) was arrived at by normalizing the [32P]dCTP incorporation in damaged plasmids against each internal control.