The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

doi:10.1016/j.mrfmmm.2016.08.004

Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis

Volumes 791–792, September–October 2016, Pages 35-41

https://doi.org/10.1016/j.mrfmmm.2016.08.004 Get rights and content

Highlights

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Human cells knockout (KO) and expressing catalytically dead (CD) variant of DNA polymerase ζ (Pol ζ) have been established by gene targeting techniques with Nalm-6 cells.
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Both Pol ζ KO and CD cells displayed prolonged cell cycle and higher incidence of micronucleus formation than the wild-type cells in the absence of exogenous genotoxic treatments.
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Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links.
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Pol ζ plays quite limited roles in protection against strand-breaks in DNA.

Abstract

Translesion DNA synthesis (TLS) is a cellular defense mechanism against genotoxins. Defects or mutations in specialized DNA polymerases (Pols) involved in TLS are believed to result in hypersensitivity to various genotoxic stresses. Here, DNA polymerase ζ (Pol ζ)-deficient (KO: knockout) and Pol ζ catalytically dead (CD) human cells were established and their sensitivity towards cytotoxic activities of various genotoxins was examined. The CD cells were engineered by altering the DNA sequence encoding two amino acids essential for the catalytic activity of Pol ζ, i.e., D2781 and D2783, to alanines. Both Pol ζ KO and CD cells displayed a prolonged cell cycle and higher incidence of micronuclei formation than the wild-type (WT) cells in the absence of exogenous genotoxic treatments, and the order of abnormality was CD > KO > WT cells. Both KO and CD cells exhibited higher sensitivity towards the killing effects of benzo[a]pyrene diol epoxide, mitomycin C, potassium bromate, N-methyl-N′-nitro-N-nitrosoguanidine, and ultraviolet C irradiation than WT cells, and there were no differences between the sensitivities of KO and CD cells. Interestingly, neither KO nor CD cells were sensitive to the cytotoxic effects of hydrogen peroxide. Since KO and CD cells displayed similar sensitivities to the genotoxins, we employed only KO cells to further examine their sensitivity to other genotoxic agents. KO cells were more sensitive to the cytotoxicity of 4-nitroquinoline N-oxide, styrene oxide, cisplatin, methyl methanesulfonate, and ethyl methanesulfonate than WT cells. However, the KO cells displayed sensitivity camptothecin, etoposide, bleomycin, hydroxyurea, crotonealdehyde, and methylglyoxal in a manner similar to the WT cells. Our results suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky DNA adducts and cross-links, but has no or limited role in the protection against strand-breaks in DNA.

Introduction

The human genome is continuously exposed to various genotoxic stresses, the origins of which can be external or internal. To circumvent this DNA damage induced by the genotoxic stresses, cells possess a variety of defense mechanisms. Translesion DNA synthesis (TLS) is a defense mechanism where specialized DNA polymerases (Pols) carry out replication of DNA across DNA damage sites [1], [2], [3]. TLS is crucial to protect the cells from DNA damage because replicative Pols are unable to continue DNA replication beyond these lesions [4], [5]. Inactivation of the specialized Pols confers hypersensitivity to genotoxic stresses [6], [7]. However, the fidelity of the specialized Pols is markedly lower than that of the replicative Pols [8], [9]. Therefore, TLS by the specialized Pols often accompanies sequence changes at or around the lesions, which, if left unrepaired, are carried forward in the next round of DNA replication [10].

Pol ζ is a specialized Pol that plays an important role in TLS [6]. Pol ζ belongs to B-family of Pols and is a heterodimer of REV3L, the catalytic subunit, and the auxiliary protein REV7, which associates with REV1 [11]. In addition, two accessory subunits of Pol δ, i.e., PolD2 and PolD3, are likely members of the active complex of Pol ζ [12]. Unlike yeast Rev3, which plays an important role in ultraviolet light (UV)-induced mutagenesis, human REV3L has a large exon encoding more than 1300 amino acids, the functions of which are not fully understood yet [12], [13], [14], [15]. Knockout (KO) of the Rev3l gene resulted in embryonic lethality in mice, and increased chromosome aberrations in embryonic fibroblasts from the KO mice [16], [17], [18]. Deficiency of REV3L in human cells prolongs cell growth and increases chromosome aberrations even in the absence of genotoxic treatments [19]. REV3L-KO in chicken DT40 cell lines increases sensitivity to the cytotoxicity of genotoxic stresses such as UV irradiation, cisplatin (CisPt), and methyl methanesulfonate (MMS) [20]. Moreover, the protective roles of Pol ζ against genotoxic stresses have also been examined with several mutagens and UV irradiation [21], [22], [23], [24], [25], [26]. However, its role has not been thoroughly investigated in human cells mainly because Pol ζ KO human cells are not easily available. In fact, the inactivation of REV3L in some of the human REV3L KO cell lines has been questioned [15], [19]. Mouse embryonic fibroblasts lacking Pol ζ are available, but are deficient in p53 functions [27]. It is reported that the mouse cells do not exhibit similar TLS efficiency and accuracy as human cells [28].

To better understand the protective roles of human Pol ζ against genotoxic stresses, we engineered REV3L-KO and catalytically dead (CD) REV3L mutant human cell lines and examined their sensitivity to cytotoxicities of 16 genotoxic chemicals and UV-C irradiation. To this end, we used human Nalm-6 cells, which possess exceptionally high gene targeting efficiencies [29], [30], [31], [32], [33]. In addition, Nalm-6 cells possess normal p53 functions and near diploid karyotype. The CD mutant of Pol ζ was generated by knock-in of a targeting sequence that directs amino acid changes of both aspartates 2781 and 2783 to alanines (D2781A, D2783A). These amino acids are responsible for the binding to magnesium ions and are essential for the catalytic activities of Pol ζ. We generated a CD mutant because we postulated that Pol ζ, in particular the middle region, might have structural roles such as interaction with other proteins [15]. If so, the results with simple KO or knock down cells may not faithfully represent the protective roles of TLS mediated by Pol ζ. In fact, the REV3L CD cells generated in this study displayed more severe phenotypes than KO cells in the absence of genotoxic stresses. Recently, a non-catalytic role has been reported for human DNA pol κ in protection against hydrogen peroxide [34]. The present results also indicated that Pol ζ protects human cells from genotoxic stresses that induce bulky DNA lesions and cross-links, but not those that induce strand breaks in DNA. Furthermore, we discuss the specificity of the protective roles of Pol ζ in human cells against endogenous and exogenous genotoxic stresses.

Section snippets

Cell culture and transfection

Nalm-6 cells were cultured in RPMI1640 supplemented with 10% calf serum, 50 μg/mL kanamycin, and 50 μM 2-mercaptoethanol at 37 °C under a 5% CO₂ atmosphere. Cells were transfected with DNA constructs by using Nucleofector I (LONZA) as previously described [35]. Transfected cells were cultured for 48 h at 37 °C and then the optimum numbers of the cells were seeded onto 96-well plates in medium containing 0.5 μg/mL puromycin (Wako Pure Chemical Industries).

Establishment of REV3L mutant cells

We have established heterozygous mutants of

Establishment of REV3L mutants

The KO cell line was generated by replacing of both alleles of exon 5 of REV3L with the drug-resistance genes (Supplementary Fig. 1A, [35]). The CD mutation was introduced into both alleles of exon 30, which resulted in the substitution of amino acids in the catalytic site of Pol, i.e., D2781A/D2783A (Supplementary Fig. 1B, [35]). Transcription of REV3L gene in KO and CD cells was analyzed by RT-PCR and DNA sequencing. We confirmed the absence of exon 5 of REV3L in the mRNA in the KO clone (

Discussion

We have engineered REV3L KO and CD cell lines and compared their phenotypes with those of WT cells. In both cell lines, the cell growth was prolonged and spontaneous micronuclei formation was increased even in the absence of exogenous genotoxic treatments, compared to the WT cells (Table 1). The disruption of Rev3l gene in mouse cells in vitro and in vivo is known to prolong the cell growth and increase the chromosome aberrations [15], [27], [36]. Our results are consistent with these reports.

Conflicts of interest statement

We have no competing interests or conflicts of interest concerning the research presented in this paper.

Acknowledgements

This work was supported by grants-in-aid for Scientific Research (A) (JSPS KAKENHI Grant 18201010 and 22241016) and (B) (JSPS KAKENHI Grant 26281029), the Ministry of Health Labour and Welfare, Japan (MHLW, H21-Food-General-009), and the Japan Health Science Foundation (KHB1007); for cancer research from MHLW (20 designated-8); the Food Safety Commission.

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DNA polymerase ζ (Pol ζ) is a specialized Pol that is involved in translesion DNA synthesis (TLS), in particular, in the extension of primer DNA after bypassing DNA lesions. Previously, we established human cells that express a variant form of Pol ζ with an amino acid change of leucine 2618 to methionine (L2618M) in the catalytic subunit REV3L (DNA Repair, 45, 34–43, 2016). This amino acid change made the cells more sensitive to the mutagenicity of benzo[a]pyrene diol epoxide (BPDE). In this study, we embedded BPDE-N²-guanine at a defined position in the supF gene on the shuttle plasmid and introduced it to REV3 L2618M cells or the wild-type (WT) cells to examine how far Pol ζ L2618M extends the primer DNA after bypassing the lesion. The adduct induced primarily G to T and G to C at the adducted site in both cell lines, but generated additional sequence changes such as base substitutions, deletions and additions in the extension patch much more often in REV3 L2618M cells than in the WT cells. Mutations in the extension patch in REV3 L2618M cells occurred most often within 10 bps from the adducted site. Then, the number of mutations gradually decreased and no mutations were observed between 30 and 40 bps from the lesion. We concluded that human Pol ζ L2618M and perhaps WT Pol ζ extend the primer DNA up to approximately 30 bps from the lesion in vivo. The possibility of involvement of Pol ζ L2618M in the insertion step of TLS is discussed.
7H-Dibenzo[c,g]carbazole: Metabolic pathways and toxicity
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The AP site is one of the most important DNA lesions causing mutagenesis/carcinogenesis due to its loss-of-base character. Additionally, AP sites might also cause large deletion mutations in living cells due to inefficient bypass of the lesion or as a byproduct of DNA repair [106]. The deletion of the whole exon 8 was identified in the V79MZh3A4 cell line after exposure to diMeDBC [58].
7H-Dibenzo[c,g]carbazole (DBC), a local and systemic carcinogen in animal studies, is a common environmental pollutant. It generally co-occurs in a variety of organic complex mixtures derived from incomplete combustion of organic matter. Despite high lipophilicity, DBC is more water-soluble and faster metabolized than the homocyclic aromatics. Moreover, greater polarity, high bioaccumulation potential, and persistence in the environment may imply DBC's higher biological significance and impact on human health, even at lower concentrations. The biotransformation pathways of DBC are incompletely known and the ultimate carcinogenic metabolite(s) are not clearly identified as yet. Structure-biological studies suggest two ways of activation: at the ring carbon atoms and at the pyrrole nitrogen. It is supposed that the particular pathway of biotransformation might be connected with the tissue/organ specificity of DBC. Cytochrome P450 (CYP) family of enzymes plays a pivotal role in the metabolism of DBC; though, the one-electron activation and the aldo-keto reductase-catalyzed oxidation are also involved in metabolic activation. Additionally, DBC can be photoactivated even at physiologically relevant doses of UVA light due to the extended aromatic ring system resulting in strong genotoxicity and oxidative stress. The goal of this review is to summarize current knowledge on mechanisms of DBC activation and possible implications for toxicity, genotoxicity, and carcinogenicity.
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In Xpa−/− MEFs transcription of two TLS polymerases was increased over that in WT: Polζ – 1.9-fold and Polκ – 3.1-fold. These results are consistent with Rev1 and Polη having roles in ICL repair [67,68], Our results may also suggest that Pol ζ plays an important role in the protection of human cells by carrying out TLS across bulky adducts (LPO adducts) and cross-links, as suggested by Suzuki and coworkers [69]. Interestingly, mRNA level of TLS polymerases appeared increased in tissues of Ercc1−/− compared to WT mice.
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Unlike DNA repair, TLS does not remove DNA lesions but simply aids the completion of DNA replication, thereby preventing DNA strand-breaks [30,31] and enhancing cell survival [22,32]. In fact, human cells lacking the catalytic activity of Pol ζ exhibit high sensitivity to the cytotoxic effects of a variety of genotoxic carcinogens [33]. However, the fidelity of DNA synthesis by specialized Pols is much lower than that of DNA synthesis by replicative Pols [34,35].
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¹: Graduate School of Biomedical & Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan.

²: Biological Safety Research Center, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.

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The role of DNA polymerase ζ in translesion synthesis across bulky DNA adducts and cross-links in human cells

Highlights

Abstract

Introduction

Section snippets

Cell culture and transfection

Establishment of REV3L mutant cells

Establishment of REV3L mutants

Discussion

Conflicts of interest statement

Acknowledgements

Adv. Protein Chem.

J. Biol. Chem.

Mutat. Res.

Curr. Biol.

Curr. Biol.

Curr. Biol.

DNA Repair

Mutat. Res.

Mutat. Res.

Biophys. Chem.

Chem. Biol.

Mutat. Res.

Mutat. Res.

J. Biol. Chem.

Cell

Chem. Biol. Interact.

Biochem. Biophys. Res. Commun.

Mutat. Res.

Mutat. Res.

Mutat. Res.

Mutat. Res.

Pharmacol. Ther.

J. Biol. Chem.

Specialized DNA polymerases, cellular survival, and the genesis of mutations

Science

DNA polymerases and cancer

Nat. Rev. Cancer

Environmental stress and lesion-bypass DNA polymerases

Annu. Rev. Microbiol.

The fidelity of DNA synthesis by eukaryotic replicative and translesion synthesis polymerases

Cell Res.

Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function

Annu. Rev. Biochem.

DNA polymerase zeta (pol ζ) in higher eukaryotes

Cell Res.

Y-family DNA polymerases in mammalian cells

Cell. Mol. Life Sci.

Error-prone DNA polymerases: when making a mistake is the only way to get ahead

Annu. Rev. Genet.

Error-prone repair DNA polymerases in prokaryotes and eukaryotes

Annu. Rev. Biochem.

Human Pol ζ purified with accessory subunits is active in translesion DNA synthesis and complements Pol η in cisplatin bypass

Proc. Natl. Acad. Sci. U. S. A.

A human homolog of the Saccharomyces cerevisiae REV3gene, which encodes the catalytic subunit of DNA polymerase ζ

Proc. Natl. Acad. Sci. U. S. A.