Formation of lysine 63-linked poly-ubiquitin chains protects human lung cells against benzo[a]pyrene-diol-epoxide-induced mutagenicity

S.A. Langie, A.M. Knaapen, C.H.M.A. Ramaekers, J. Theys, J. Brun, R.W. Godschalk, F.J. van Schooten, P. Lambin, D.A. Gray, B.G. Wouters, R.K. Chiu*

*Corresponding author for this work

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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 languageEnglish
Pages (from-to)852-862
Number of pages11
JournalDna Repair
Volume6
Issue number6
DOIs
Publication statusPublished - 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

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