DNA Polymerases In Mutagenesis Research Articles

Opposing roles of Y-family DNA polymerases in lipid peroxide mutagenesis at the hisG46 target in the Ames test

DNA polymerases play a key role in mutagenesis by performing translesion DNA synthesis (TLS). The Y-family of DNA polymerases comprises several evolutionarily conserved families, specializing in TLS of different DNA adducts. Exocyclic etheno and propano DNA adducts are among the most common endogenous DNA lesions induced by lipid peroxidation reactions triggered by oxidative stress. We have investigated the participation of two enterobacterial representatives of the PolIV and PolV branches of Y-family DNA polymerases in mutagenesis by two model lipid peroxidation derived genotoxins, glyoxal and crotonaldehyde. Mutagenesis by the ethano adduct (glyoxal-derived) and the propano adduct (crontonaldehyde-derived) at the GC target in the Ames test depended exclusively on PolV type DNA polymerases such as PolRI. In contrast, PolIV suppressed glyoxal and, even more, crotonaldehyde mutagenesis, as detected by enzyme overexpression and gene knockout approaches. We propose that DNA polymerase IV, which is the mammalian DNA polymerase κ ortholog, acts as a housekeeper protecting the genome from lipoxidative stress.

Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo.

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc.

Roles of specialized DNA polymerases in mutagenesis by 8-hydroxyguanine in human cells

The formation of 8-hydroxyguanine (8-OH-Gua, 7,8-dihydro-8-oxoguanine) in DNA and in the nucleotide pool results in G:C-->T:A and A:T-->C:G substitution mutations, respectively, due to the ability of 8-OH-Gua to pair with both C and A. In this study, shuttle plasmid DNAs containing 8-OH-Gua paired with C and A in the supF gene were transfected into human 293T cells, in which specialized DNA polymerases were knocked-down. The DNAs replicated in the cells were recovered and then introduced into an indicator strain of Escherichia coli. Mutation analysis indicated that the knock-downs of DNA polymerases eta and zeta by siRNAs enhanced the G:C-->T:A mutations caused by 8-OH-Gua:C. The 8-OH-Gua:C-induced mutation frequency was not further increased by double knock-downs of DNA polymerases eta and zeta, suggesting that the two DNA polymerases work in the same pathway. In addition, the reduction of DNA polymerase eta slightly decreased the A:T-->C:G substitutions caused by 8-OH-Gua:A. These results suggest that DNA polymerases eta and zeta are involved in the bypass of 8-OH-Gua in human cells.

Roles of specialized DNA polymerases in mutagenesis by oxidized guanine

To assess the involvement of specialized DNA polymerases in the mutagenesis induced by an oxidized form of dGTP, 8-hydroxy-2'-deoxyguanosine 5'-triphosphate (8-OH-dGTP, 7,8-dihydro-8-oxo-2'-deoxyguanosine 5'-triphosphate), shuttle plasmid DNA containing the supF gene and 8-OH-dGTP were introduced into human 293T cells in which specialized DNA polymerases were knocked-down by siRNAs. The knock-downs of DNA polymerases eta and zeta, and REV1 reduced the A:T-->C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A. In contrast, the knock-down of DNA polymerase tau did not affect the mutations induced by 8-OH-dGTP. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols eta plus zeta and REV1 plus DNA pol zeta (but not by that of DNA pol eta plus REV1), suggesting that REV1-DNA pol eta and DNA pol zeta work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by 8-OH-dGTP.

Involvement of specialized DNA polymerases in mutagenesis by 8-hydroxy-dGTP in human cells

The mutagenicity of an oxidized form of dGTP, 8-hydroxy-2′-deoxyguanosine 5′-triphosphate (8-OH-dGTP), was examined using human 293T cells. Shuttle plasmid DNA containing the supF gene was first transfected into the cells, and then 8-OH-dGTP was introduced by means of osmotic pressure. The DNAs replicated in the cells were recovered and then transfected into Escherichia coli. 8-OH-dGTP induced A:T → C:G substitution mutations in the cells. The knock-downs of DNA polymerases η and ζ, and REV1 by siRNAs reduced the A:T → C:G substitution mutations, suggesting that these DNA polymerases are involved in the misincorporation of 8-OH-dGTP opposite A in human cells. In contrast, the knock-down of DNA polymerase ι did not affect the 8-OH-dGTP-induced mutations. The decrease in the induced mutation frequency was more evident by double knock-downs of DNA pols η plus ζ and REV1 plus DNA pol ζ (but not by that of DNA pol η plus REV1), suggesting that REV1-DNA pol η and DNA pol ζ work in different steps. These results indicate that specialized DNA polymerases are involved in the mutagenesis induced by the oxidized dGTP.

Novel DNA Polymerases and Novel Genotoxicity Assays

Human genome is continuously exposed to exogenous and endogenous genotoxic agents. The most hazardous and ubiquitous exogenous mutagen may be cigarette smoke, which contains more than 4,000 chemicals including about 60 known carcinogens. About 1% of oxygen metabolism leads to production of reactive oxygen species, a major source of endogenous mutagens. These genotoxic agents induce a variety of lesions in DNA, which results in mutations and chromosome aberrations upon replication. If such genetic alterations occurred in the genes involved in cell proliferations and/or maintenance of genome stability, the cells would proceed in multi-steps of carcinogenesis. The goal of environmental mutagenesis and genetic toxicology is to elucidate the mechanistic links between exposure to genotoxic agents and the health consequences, and to prevent the health hazard associated with DNA damage. To this end, we have investigated the mechanisms of mutagenesis induced by environmental chemicals and contributed to establish the paradigm that Y-family DNA polymerases play central roles in mutagenesis via translesion DNA synthesis across damaged bases in DNA. We also developed genotoxicity assays with bacteria and mice to evaluate the potential risk of environmental chemicals. Here, I review the roles of Y-family DNA polymerases in mutagenesis and introduce features of the novel bacterial and rodent genotoxicity assays. Future directions of environmental mutagenesis and carcinogenesis are also discussed.

Environmental Stress and Lesion-Bypass DNA Polymerases

In nature, microbes live under a variety of harsh conditions, such as excess DNA damage, starvation, pH shift, or high temperatures. Microbial cells respond to such stressful conditions mostly by switching global patterns of gene expression to relieve the environmental stress. The SOS response, which is induced by DNA damage, is one such global network of gene expression that plays a crucial role in balancing the genomic stability and flexibility that are necessary to adapt to harsh environments. Here, I review the roles of SOS-inducible and noninducible lesion-bypass DNA polymerases in mutagenesis induced by environmental stress, and discuss how these polymerases are coordinated for the replication of damaged chromosomes. Possible contributions of lesion-bypass DNA polymerase in hyperthermophilic archaea, e.g., Sulfolobus solfataricus, to genome maintenance are also discussed.

Involvement of Y-Family DNA Polymerases in Mutagenesis Caused by Oxidized Nucleotides in Escherichia coli

Escherichia coli DNA polymerase IV incorporated 2-hydroxy-dATP opposite template guanine or thymine and 8-hydroxy-dGTP exclusively opposite adenine in vitro. Mutator phenotypes in sod/fur strains were substantially diminished by deletion of dinB and/or umuDC. DNA polymerases IV and V may be involved in mutagenesis caused by incorporation of the oxidized deoxynucleoside triphosphates.

Oxygen radical induced mutagenesis is DNA polymerase specific

Oxygen free radicals are produced in large amounts by normal cellular processes. Damage to DNA by these reactive species has been implicated in mutagenesis and may be important in the etiology of a variety of human diseases. In this study we investigate the types of mutations produced in vitro as a result of DNA damage by oxygen free radicals. We used a lacZ alpha forward mutation assay in which M13 viral DNA is damaged in vitro, replicated with purified DNA polymerase alpha or beta, transfected into E. coli, and screened for mutations by reduced alpha-complementation of beta-galactosidase activity. By determining the effects of damaged templates on the fidelity of individual DNA polymerases involved in replication and repair, we address the role of specific DNA polymerases in mutagenesis induced by reactive oxygen species. Aerobic incubation of DNA with 100 microM CuCl, 10 microM H2O2 and 100 microM ascorbic acid results in a 3.3-fold and a 3.6-fold elevation in mutation frequency for polymerases alpha and beta, respectively. The specificity and location of the induced mutations, however, are entirely different. For polymerase alpha, A to C, and C to A transversions and deletions of C are each elevated more than 10-fold over their frequencies on undamaged template. For polymerase beta, A to T, C to T, C to A, G to C, and G to T substitutions, and deletions of G are elevated by damage. The frequency of mutants containing two or more closely spaced substitutions is also markedly increased by template damage although the types of mutations and their positions are again specific to each DNA polymerase. We conclude that, for oxidative lesions, the frequency and the types of mutations are determined in part by the DNA polymerase that encounters the site of damage.