Abstract
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 polkappa and poleta in the BPDE-induced S-phase checkpoint. BPDE treatment induced the re-localization of an ectopically expressed green fluorescent protein-polkappa 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-poleta 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-polkappa 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 poleta 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 gammaH2AX (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 polkappa, replication forks stall at sites of damage and collapse and generate DSBs. Therefore, we conclude that the trans-lesion synthesis enzyme polkappa is specifically required for normal recovery from the BPDE-induced S-phase checkpoint.
Highlights
DNA damage, mutagenesis, and carcinogenesis [1]
benzo[a]pyrene-dihydrodiol epoxide (BPDE) Induces Formation of GFP-pol Nuclear Foci at Sites of Ongoing DNA Synthesis—Other workers have shown [30, 36] that a fusion protein of pol with GFP is recruited to discrete nuclear foci in response to genotoxins
In this report we have investigated the roles of the TLS enzymes pol and pol in the S-phase checkpoint elicited by the carcinogen BPDE
Summary
DNA damage, mutagenesis, and carcinogenesis [1]. The mechanism of B[a]P-induced carcinogenesis has been studied extensively and is well understood [4]. It is widely hypothesized that cell cycle checkpoints are important tumor-suppressive mechanisms that contribute to the maintenance of genomic stability. Consistent with this hypothesis, individuals with congenital defects in checkpoint genes (e.g. p53, CHK2, and ATM) show increased propensity to tumorigenesis. Bulky DNA lesions (such as UV light-induced pyrimidine dimers or BPDE adducts) or replication stress (resulting from depletion of nucleotide pools) elicits a checkpoint pathway mediated by the ATM-related kinase ATR and its downstream effector kinase Chk1 [6]. The mechanism of Chk1-mediated S-phase arrest is less clear but might involve degradation of the Cdc25A protein phosphatase [15, 16] and inhibition of Cdc7-Dbf kinase complex [17]
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