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

Sabine A S Langie, Ad M Knaapen, Chantal H M A Ramaekers, Jan Theys, Jan Brun, Roger W L Godschalk, Frederik J van Schooten, Philippe Lambin, Douglas A Gray, Bradly G Wouters, Roland K Chiu

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9 Citations (Scopus)


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. POLeta), 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 (WT-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 POLeta to the replication machinery of the K63R-Ub expressing cells, where it co-localized with PCNA. Suppression of POLeta 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)852-62
Number of pages11
JournalDna repair
Issue number6
Publication statusPublished - 1-Jun-2007
Externally publishedYes


  • Benzo(a)pyrene
  • Cell Line, Tumor
  • DNA Damage
  • DNA Mutational Analysis
  • DNA Repair
  • Dose-Response Relationship, Drug
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Lung
  • Lung Neoplasms
  • Lysine
  • Mutagens
  • Polyubiquitin
  • Ultraviolet Rays
  • Journal Article
  • Research Support, Non-U.S. Gov't

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