Abstract
Benzo[a]pyrene exerts its mutagenic effects via induction of benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts. Such helix- distorting adducts are not always successfully repaired prior to DNA replication, which may result in a blocked replication fork. To alleviate this stall, cells utilize DNA damage tolerance systems involving either error-free damage avoidance or error-prone translesion synthesis. Studies in yeast suggest the modification of PCNA by lysine 63-linked poly-ubiquitin (K63-polyUb) chains as a key mediator of the error-free damage avoidance pathway Recently, we extended this observation to human cells, showing the occurrence of poly-ubiquitination of PCNA in UV-irradiated human cells. in the present study, we hypothesized that disrupting the formation of K63-polyUb chains inhibits damage avoidance and favors error-prone repair involving low-fidelity polymerases (e.g. POLq), causing increased BPDE-induced mutagenicity To test this hypothesis, we generated A549 cells expressing either a mutant ubiquitin (K63R-Ub) which blocks further ubiquitination through K63, or the wild type ubiquitin (VVT-Ub). We show that PCNA is poly- ubiquitinated in these cells upon BPDE-exposure and that disruption of K63-polyUb chain formation has no effect on BPDE-induced toxicity In contrast, significantly higher frequencies of BPDE-induced HPRT mutations were observed in K63R-Ub expressing cells, of which the majority (74%) was G - T transversion. BPDE treatment caused an enhanced recruitment of POL'q to the replication machinery of the K63R-Ub expressing cells, where it co-localized with PCNA. Suppression of POLq expression by using siRNA resulted in a 50% reduction of BPDE-induced mutations in the K63R cells. In conclusion, we demonstrated that formation of K63-polyUb chains protects BPDE-exposed human cells against translesion synthesis -mediated mutagenesis. These findings indicate that K63-polyubiquitination guards against chemical carcinogenesis by preventing mutagenesis and thus contributing to genomic stability.
Original language | English |
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Pages (from-to) | 852-862 |
Number of pages | 11 |
Journal | Dna Repair |
Volume | 6 |
Issue number | 6 |
DOIs | |
Publication status | Published - 1 Jun 2007 |
Keywords
- benzo(a)pyrene
- DNA damage tolerance
- mutagenesis
- translesion synthesis
- ubiquitin
- DNA-POLYMERASE-KAPPA
- WHITE BLOOD-CELLS
- DIOL EPOXIDE
- EXCISION-REPAIR
- TRANSLESION SYNTHESIS
- CONJUGATING ENZYMES
- MAMMALIAN-CELLS
- ADDUCTS
- PCNA
- REPLICATION