FIG. 9.

Roles of RPA, ATR, and Chk1 in BPDE-induced PCNA ubiquitination and Polκ recruitment. (A) H1299 cells were transfected with siRNA oligonucleotide duplexes against ATR and RPA or with control Cy3 RNA oligonucleotides. Some cultures were infected with AdChk1KR or AdCon. The resulting cells were analyzed for ATR, RPA, or Chk1 expression with appropriate antibodies. (B) H1299 cells expressing GFP-Polκ were transfected with siRNA oligonucleotides against ATR, RPA, and Rad18 or with Cy3 (control) oligonucleotides. Alternatively, some cultures were infected with AdChk1KR. The resulting cultures were given 600 nM BPDE, and 6 h later the cells were lysed. Nuclear fractions were immunoprecipitated with anti-PCNA, and the resulting immune complexes were resolved by SDS-PAGE and immunoblotted (IB) with anti-PCNA or anti-GFP antibodies. (C) ET163 and YZ5 cells were treated with 600 nM BPDE or left untreated as controls. After 4 h, soluble and chromatin fractions were prepared and analyzed directly for PCNA (chromatin fraction) and Thr68-phosphorylated Chk2 (soluble fraction) levels.