title: Formation of lysine 63-linked poly-ubiquitin chains protects human lung cells against benzo[a]pyrene-diol-epoxide-induced mutagenicity.
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. 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.
authors: Department of Health Risk Analysis and Toxicology NUTRIM, Maastricht University, Maastricht, The Netherlands.
authors: Sabine A S
authors: SA
authors: Langie
authors: Ad M
authors: AM
authors: Knaapen
authors: Chantal H M A
authors: CH
authors: Ramaekers
authors: Jan
authors: J
authors: Theys
authors: Jan
authors: J
authors: Brun
authors: Roger W L
authors: RW
authors: Godschalk
authors: Frederik J
authors: FJ
authors: van Schooten
authors: Philippe
authors: P
authors: Lambin
authors: Douglas A
authors: DA
authors: Gray
authors: Bradly G
authors: BG
authors: Wouters
authors: Roland K
authors: RK
authors: Chiu
mesh_terms: D001564:Benzo(a)pyrene / Q000737:chemistry*; D045744:Cell Line, Tumor; D004249:DNA Damage; D004252:DNA Mutational Analysis; D004260:DNA Repair; D004305:Dose-Response Relationship, Drug; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008168:Lung / Q000187:drug effects*; D008175:Lung Neoplasms / Q000139:chemically induced* / Q000517:prevention & control*; D008239:Lysine / Q000737:chemistry*; D009153:Mutagens; D025821:Polyubiquitin / Q000737:chemistry* / Q000378:metabolism; D014466:Ultraviolet Rays
chemical_list: D009153:Mutagens; D025821:Polyubiquitin; D049452:Green Fluorescent Proteins; D001564:Benzo(a)pyrene; D008239:Lysine