DNA polymerase κ is specifically required for recovery from the benzo[a]pyrene-dihydrodiol epoxide (BPDE)-induced S-phase checkpoint

Previously we identified an intra-S-phase cell cycle checkpoint elicited by the DNA-damaging carcinogen benzo[ a] pyrene-dihydrodiol epoxide ( BPDE). Here we have investigated the roles of lesion bypass DNA polymerases pol kappa and pol eta in the BPDE- induced S-phase checkpoint. BPDE treatment induced the re-localization of an ectopically expressed green fluorescent protein-pol kappa fusion protein to nuclear foci containing sites of active DNA synthesis in human lung carcinoma H1299 cells. In contrast, a similarly expressed yellow fluorescent protein-pol eta fusion protein showed a constitutive nuclear focal distribution at replication forks ( in the same cells) that was unchanged in response to BPDE. BPDE- induced formation of green fluorescent protein-pol kappa nuclear foci was temporally coincident with checkpoint-mediated S-phase arrest. Unlike wild-type cells, Polk(-/-) mouse embryonic fibroblasts (MEFs) failed to recover from BPDE- induced S-phase arrest, while exhibiting normal recovery from S-phase arrest induced by ionizing radiation and hydroxyurea. XPV fibroblasts lacking pol eta showed a normal S-phase checkpoint response to BPDE ( but failed to recover from the UV light-induced S-phase checkpoint), in sharp contrast to Polk(-/-) MEFs. The persistent S-phase arrest in BPDE-treated PolK(-/-) cells was associated with increased levels of histone gamma H2AX ( a marker of DNA double- strand breaks (DSBs)) and activation of the DSB-responsive kinases ATM and Chk2. These data suggest that in the absence of pol kappa, replication forks stall at sites of damage and collapse and generate DSBs. Therefore, we conclude that the trans-lesion synthesis enzyme pol kappa is specifically required for normal recovery from the BPDE- induced S- phase checkpoint.