molecular mechanism

Fundamental and Molecular Mechanism of Mutagenesis
vol. 546, issue. 1-2. 26 Feb.2004
Phosphorylation and nuclear accumulation are distinct events contributing to the activation of p53
Heather M. O'Hagana and Mats Ljungman ,  , a, b

a Department of Radiation Oncology, Division of Radiation and Cancer Biology, Program in Molecular and Cellular Biology, University of Michigan Medical School, Ann Arbor, MI 48109-0936, USA
b Department of Environmental Health Sciences, School of Public Health, University of Michigan, Ann Arbor, MI 48109-0936, USA

Received 25 March 2003;  revised 8 October 2003;  accepted 14 October 2003. ; Available online 5 December 2003.

Abstract
It has been recently shown that ionizing radiation (IR) and the mRNA synthesis inhibitor 5,6-dichloro-1-b- -ribofuranosylbenzimidazole (DRB) act in synergy to induce p53-mediated transactivation of reporter plasmids in human cells [Oncogene 19 (2000) 3829]. We have extended these studies and show that ionizing radiation and DRB also act in synergy to induce ATM-mediated phosphorylation of the ser15 site of p53 and enhance the expression of endogenous p21 protein. Examination of the localization of p53 revealed that while DRB did not induce phosphorylation of the ser15 site of p53 but efficiently accumulated p53 in the nucleus, ionizing radiation induced phosphorylation of the ser15 site of p53 without prolonged nuclear accumulation. Importantly, the combination of DRB and IR resulted in a strong accumulation of phosphorylated p53 in the nucleus that was more persistent then p53 accumulation after IR alone. Furthermore, the nuclear export inhibitor leptomycin B showed a similar synergy with IR as did DRB regarding ser15 phosphorylation of p53 and p21 induction. These results suggest that the synergistic activation of the p53 response by the combination treatment is due to the activation of two distinct pathways where DRB causes the prolonged nuclear accumulation of p53 while ionizing radiation activates p53 by ATM-mediated phosphorylation.
Author Keywords: Ionizing radiation; DRB; Inhibition of transcription; ATM; Leptomycin B


Developing azo and formazan dyes based on environmental considerations: Salmonella mutagenicity
Laura C. Edwardsa, Harold S. Freeman ,  , a and Larry D. Claxtonb

a Department of Textile Chemistry, College of Textiles, North Carolina State University, Raleigh, NC 27695-8301, USA
b U.S. EPA, National Health and Environmental Effects Research Laboratory, Research Triangle Park, NC 27709, USA

Received 28 May 2003;  revised 18 September 2003;  accepted 14 October 2003. ; Available online 9 December 2003.

Abstract
In previous papers, the synthesis and chemical properties of iron-complexed azo and formazan dyes were reported. It was shown that in certain cases iron could be substituted for the traditionally used metals such as chromium and cobalt, without having an adverse effect on dye stability. While these results suggested that the iron analogs were potential replacements for the commercially used chromium and cobalt prototypes, characterization of potentially adverse environmental effects of the new dyes was deemed an essential step in their further development. The present paper provides results from using the Salmonella/mammalian microsome assay to determine the mutagenicity of some important commercial metal complexed dyes, their unmetallized forms, and the corresponding iron-complexed analogs. The study compared the mutagenic properties of six unmetallized azo dyes, six commercial cobalt- or chromium-complexed azo dyes, six iron-complexed azo dyes, six unmetallized formazan dyes, and six iron-complexed formazan dyes. The results of this study suggest that the mutagenicity of the unmetallized dye precursors plays a role in determining the mutagenicity of the iron-complexes. For the monoazo dye containing a nitro group, metal complex formation using iron or chromium decreased or removed mutagenicity in TA100; however, little reduction in mutagenicity was noted in TA98. For the formazan dye containing a nitro group, metal-complex formation using iron increased mutagenicity. Results varied for metal-complexes of azo and formazan dyes without nitro groups, but in general, the metal-complexed dyes based on mutagenic ligands were also mutagenic, while those dyes based on nonmutagenic ligands were nonmutagenic.
Author Keywords: Salmonella; Metal-complex; Formazan dye



Effects of oxidative and alkylating damage on microsatellite instability in nontumorigenic human cells
Mandy L. Maneval and Kristin A. Eckert ,

MD/PhD Training Program, Jake Gittlen Cancer Research Institute, Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA

Received 23 July 2003;  revised 7 October 2003;  accepted 14 October 2003. ; Available online 5 December 2003.

Abstract
Microsatellite instability is a phenomenon that is well characterized in mismatch repair-deficient tumor cell lines, including the potential etiological role of endogenous DNA damage. However, our understanding of microsatellite mutational mechanisms in repair-proficient, nontumorigenic cells is limited. We determined microsatellite mutation frequencies for human lymphoblastoid cells using an episomal DNA shuttle vector in which a (TTCC/AAGG)9 microsatellite is inserted in-frame within the herpes simplex virus thymidine kinase (HSV-tk) gene. The responses of plasmid-bearing cells to reactive oxygen species or alkylating agents were compared after treatment with hydrogen peroxide (H2O2) and N-ethyl-N-nitrosourea (ENU). H2O2 treatment induced a statistically significant increase in overall HSV-tk mutation frequency relative to controls, with catalase reducing the effect. H2O2 treatment increased the mutation frequency within the microsatellite and the HSV-tk coding region to a similar extent (five and six-fold, respectively, relative to the control). Mutational specificity analyses demonstrated that the proportion of mutations within the microsatellite is not statistically different among the H2O2, catalase, and PBS treatment groups. In contrast, treatment of cells bearing the microsatellite vector with ENU altered the mutational spectrum, relative to solvent control. ENU induced the expected base substitutions within the HSV-tk coding region, but did not increase the microsatellite mutation frequency. The low level of microsatellite mutagenesis observed after reactive oxygen species (ROS) insult likely reflects the normal repair processes of these nontumorigenic, repair-competent cells. Our ex vivo experiments demonstrate the manner in which repetitive DNA in normal human cells might respond to endogenous mutagens.
Author Keywords: Herpes simplex virus thymidine kinase; Microsatellite instability; Oxidative; Damage; Hydrogen peroxide
Abbreviations: FUdR, 5-fluoro-2'-deoxyuridine; HSV-tk, herpes simplex virus type 1 thymidine kinase; MSI, microsatellite instability; H2O2, hydrogen peroxide; ENU, N-ethyl-N-nitrosurea; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline

Cell death evaluation in benzo[a]pyrene-transformed human breast epithelial cells after microcell-mediated transfer of chromosomes 11 and 17
Maria Luiza S. Mello ,  , a, Luis Fernando Barbisanb, Mohamed H. Lareefc, Jose Russoc and Benedicto de Campos Vidala

a Department of Cell Biology, Institute of Biology, UNICAMP, 13084-971, Campinas (SP), Brazil
b Department of Morphology, Institute of Biosciences, UNESP, 18618-000, Botucatu (SP), Brazil
c Fox Chase Cancer Center, Philadelphia, PA 19111, USA

Received 2 May 2003;  revised 19 September 2003;  accepted 15 October 2003. ; Available online 9 December 2003.

Abstract
The incidence of apoptosis and nuclear instability, including the incidence of catastrophic death, were investigated in benzo[a]pyrene (BP)-transformed human breast epithelial cells (BP1-E cell line) after microcell-mediated transfer of chromosomes 11 and 17. Since the introduction of normal chromosomes 11 and 17 into tumorigenic human breast BP1-E cells reverts some of these cells' characteristics (especially those affected by microsatellite instabilities and loss of heterozygosity) to those of parental non-transformed MCF-10F cells, it was expected that the cell death rates would also be affected by this treatment. The transfer of the mentioned chromosomes, especially chromosome 17, to tumorigenic BP1-E cells increased the apoptotic ratios and decreased the nuclear instability ratios, thus showing that the microsatellite instability and loss of heterozygosity induced by BP in these chromosomes of MCF-10F cells affect the control of cell death mechanisms.
Author Keywords: Apoptosis; Catastrophic death; Human breast epithelial cells; Chromosome 11; Chromosome 17





Evidence of association of the CYP2E1 genetic polymorphism with micronuclei frequency in human peripheral blood
Hitoshi Ishikawa ,  , a, Hidetaka Yamamotob, Ying Tiana, d, Mitsuo Kawanoc, Toru Yamauchia and Kazuhito Yokoyamaa

a Department of Public Health and Preventive Medicine, Mie University School of Medicine, Edobashi 2-174, Tsu 514-8507, Japan
b Department of Forensic Medicine and Sciences, Mie University School of Medicine, Edobashi 2-174, Tsu 514-8507, Japan
c Department of Microbiology, Mie University School of Medicine, Edobashi 2-174, Tsu 514-8507, Japan
d School of Public Health, Shanghai Second Medical University, No. 280, ChongQing Nan Road, Shanghai, 200025, PR China

Received 31 May 2003;  revised 21 October 2003;  accepted 31 October 2003. ; Available online 19 December 2003.

Abstract
Micronuclei (MN) are used as one of the cytogenetic biomarkers, and intra- and inter-individual variations in this frequency have been reported in human blood lymphocytes. Polymorphisms in a few metabolic enzyme genes seem to account for a proportion of this variability, but the impacts of specific genetic variants on the MN frequency have not yet been clarified. Here, we investigated the relationship between the MN frequency and several gene polymorphisms in 90 healthy Japanese men. The subjects with the CYP2E1*3 variant allele had a statistically lower mean MN frequency than subjects with the CYP2E1*1/*1 wild type. Furthermore, the adjusted odds ratio (OR) of the CYP2E1*3 variant with higher MN frequency levels was also significantly lower and calculated to be 0.25 (95% CI 0.07–0.83), when the OR for the subjects with the CYP2E1*1/*1 wild type was defined as 1.00. These data suggest that the CYP2E1*3 polymorphism may have the potential to influence the baseline frequency of MN.
Author Keywords: Age; Alcohol; CYP2E1 polymorphism; Micronuclei; Odds ratio; RsaI
Abbreviations: CAs, chromosomal aberrations; SCEs, sister chromatids exchanges; MN, micronucleus/micronuclei; CYP1A1, cytochrome p450 1A1; CYP2E1, cytochrome p450 2E1; GSTM1, glutathione S-transferase M1; GSTT1, glutathione S-transferase T1; OR, odds ratio; CI, confidence intervals


Aphidicolin and bleomycin induced chromosome damage as biomarker of mutagen sensitivity: a twin study
Bruna Tedeschi ,  , a, Rosadele Cicchettia, Gabriella Argentina, Daniela Caporossia, Monica Pittalugab, Paolo Parisib and Patrizia Vernolea

a Department of Public Health and Cell Biology, University of Rome "Tor Vergata", via Montpellier 1, 0013, Rome, Italy
b Human Biology Center, University Institute of Movement Science (IUSM), Rome, Italy

Received 9 June 2003;  revised 24 October 2003;  accepted 31 October 2003. ; Available online 13 December 2003.

Abstract
The induction of chromosome damage in cultured human lymphocytes by in vitro treatments with aphidicolin (APC) and bleomycin (BLM) has been proposed as test of sensitivity to mutagens. To assess their validity, we have investigated whether the individual expression of induced chromosome damage has a genetic rather than an environmental basis. Metaphase analysis for chromosomal aberrations (CA) and micronucleus (MN) assay in cytokinesis-blocked cells have been performed in peripheral blood lymphocytes from 19 healthy male twins (9 monozygotic and 10 dizygotic pairs), aged 70–78 years, after APC, BLM and APC+BLM treatments.
Concordance between twins revealed a high genetic component in the sensitivity towards clastogenic action of APC both as percentages of CA and MN. The micronucleus assay demonstrated a genetic basis also in the expression of chromosome damage induced by BLM and APC+BLM treatments. Since twins were elderly people, to investigate the possible role of age, CA and MN frequencies were compared with those found in lymphocytes from 11 young male donors. Basal and APC-induced chromosome damage were clearly increased in the former. Following BLM and APC+BLM treatments, age significantly increased mitotic delay, as shown by the mitotic indexes (MI) and by the ratios between binucleated and mononucleated (B/M) cells.
Author Keywords: Human twins; Age; Chromosome damage; Bleomycin; Aphidicolin




Two regions in chromosome 19q13.2-3 are associated with risk of lung cancer
Ulla Vogel ,  , a, Imke Larosb, Nicklas R. Jacobsena, Birthe L. Thomsenc, Helle Bakc, Anja Olsenc, Zuzanna Bukowyb, Håkan Wallina, Kim Overvadd, Anne Tjønnelandc, Bjørn A. Nexøb and Ole Raaschou-Nielsenc

a National Institute of Occupational Health, DK-2100, Copenhagen O, Denmark
b Institute of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000, Aarhus C, Denmark
c Institute of Cancer Epidemiology, The Danish Cancer Society, DK-2100, Copenhagen, Denmark
d Department of Clinical Epidemiology, Aalborg Hospital and Aarhus University Hospital, and Department of Epidemiology and Social Medicine, University of Aarhus, DK-8000, Aarhus C, Denmark

Received 1 September 2003;  revised 26 September 2003;  accepted 7 November 2003. ; Available online 18 December 2003.

Abstract
Lung cancer is the most common fatal cancer among Danish men, and the incidence rate is increasing among women. In a case-cohort study, we have investigated the occurrence of lung cancer in relation to a high-risk haplotype, previously identified for breast cancer among post-menopausal women, and in relation to the closely linked polymorphisms XPD Asp312Asn and Lys751Gln. Among 54,220 members of a Danish prospective cohort study aged 50–64 at entry, 265 lung cancer cases were identified and a sub-cohort comprising 272 individuals was used for comparison. Among women in the 50–55 year age interval, homozygous carriers of the high-risk haplotype were at increased risk of lung cancer (RR=7.02, 95% CI=1.88–26.18). In the 56–60 year and 61–70 year age intervals, no associations were observed. Among men, no statistically significant associations were found in any age interval. Female homozygous carriers of the variant allele of XPD Lys751Gln were at significantly increased risk of lung cancer in the two younger age-intervals (50–55 years: RR=5.60, 95% CI=1.18–26.45, 56–60 years: RR=10.60, 95% CI=1.50–75.64). Among men, carriers of the variant allele of XPD Lys751Gln had a non-significantly increased risk of lung cancer in the youngest age interval (RR=6.38, 95% CI=0.74–54.90). When the polymorphisms in XPD Asp312Asn and Lys751Gln were mutually adjusted, XPD Asp312Asn was not associated with increased risk of cancer. We found no interaction between genotypes and duration of smoking. In conclusion, two regions of chromosome 19q13.2-3 seem to be associated with risk of lung cancer.
Author Keywords: XPD; RAI; ERCC1; ASE-1; DNA repair; Polymorphism
Abbreviations: CI, confidence interval; RR, rate ratio; SNP, single nucleotide polymorphism

Identification of paraoxonase 3 gene (PON3) missense mutations in a population of southern Italy
Salvatore Campo ,  , a, Adriana M. Sardob, Giuseppe M. Campoa, Angela Avenosoa, Maria Castaldob, Angela D'Ascolaa, Elena Giuntac, Alberto Calatronia and Antonino Saittab

a Department of Biochemical, Physiological and Nutritional Sciences, School of Medicine, University of Messina, Policlinico Universitario, Torre Biologica, 5° Piano, Via C. Valeria 1, Messina 98125, Italy
b Department of Internal Medicine, School of Medicine, University of Messina, Policlinico Universitario, Messina 98125, Italy
c "Ospedale Pappardo", Contrada Sperone, Messina 98100, Italy

Received 8 June 2003;  revised 11 November 2003;  accepted 25 November 2003. ; Available online 24 January 2004.

Abstract
PON gene family includes at least three members termed PON1, PON2 and PON3, and it is mapped on human chromosome 7q21–q22. PON1 and PON3 gene products are constituents of high density lipoprotein (HDL) and have many enzymatic properties and antioxidant activity. PONs are proposed to participate in the prevention of low density lipoprotein (LDL) oxidation. PON1 and PON2 genes have missense polymorphisms, but, to date, no missense variants are reported in PON3 gene. In this work we explored the existence of genetic variants within the PON3 coding sequences. Five point mutations were identified by direct sequencing of genomic DNA derived from 250 randomly selected DNA samples of 1143 blood donors living in southern Italy. Three were silent mutations, while two were missense mutations that give rise to amino acid substitutions at positions 311 (S>T) and 324 (G>D). The missense variations in the DNA of the 1143 samples had frequencies of 0.22% (5 out of 2286 alleles) for the S311T mutation, and 0.57% (13 out of 2286 alleles) for the G324D mutation. The effect of these variants on the metabolic activity of paraoxonase 3 remains to be further evaluated.
Author Keywords: Paraoxonase 3; Lipid peroxidation; Antioxidants; Atherosclerosis; Missense mutation




Mutagenicity of cadmium in mammalian cells: implication of oxidative DNA damage
Metka Filipi  ,  , a and Tom K. Heib

a Department of Genetic Toxicology and Cancer Biology, National Institute of Biology, Ve na pot 111, 1000, Ljubljana, Slovenia
b Center for Radiological Research, College of Physicians and Surgeons, and Department of Environmental Health Sciences, Joseph Mailman School of Public Health, Columbia University, 630 West 168th Street, New York, NY 10032, USA

Received 19 July 2003;  revised 11 November 2003;  accepted 25 November 2003. ; Available online 24 January 2004.

Abstract
Cadmium and cadmium compounds are well established human carcinogens and are ubiquitously present in the environment. The carcinogenic mechanism(s) of cadmium remains largely unknown since direct mutagenic effect is weak in bacterial and in standard mammalian cell mutation assays. In this study, we show that when evaluated using the human-hamster hybrid AL cell mutation assay in which both intragenic and multilocus deletions can readily be detected, CdCl2 is a strong mutagen that induces predominantly large deletion mutations. Concurrent treatment of AL cells with the oxyradical scavenger dimethyl sulfoxide significantly reduced the number of cadmium-induced mutations. In contrast, pre-treatment of cells with buthionine sulfoximine that depletes intracellular glutathione, increased cytotoxicity and mutagenicity of cadmium. These results demonstrate that reactive oxygen species mediate cadmium induced mutations in AL cells. With laser scanning confocal microscopy and the fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, we demonstrated that cadmium induced a dose and time dependent formation of intracellular oxyradicals. Using immunoperoxidase staining coupled with a monoclonal antibody-specific for 8-OHdG adducts in DNA, we demonstrated that cadmium induced a dose dependent increase of 8-OHdG adducts, which accumulated with prolonged exposure. Furthermore, we showed that at low concentration, cadmium, attenuated removal of hydrogen peroxide induced 8-OHdG adducts. Thus, the carcinogenicity of cadmium can, in part, be explained by its mutagenic activity, which is mediated by reactive oxygen species induced DNA damage and by its interference with the repair of oxidative DNA damage.
Author Keywords: Cadmium; AL mutation assay; Mutational spectrum; Reactive oxygen species; 8-OHdG; DNA repair



Genoprotective pathways
Part I. Extracellular signaling through Gs protein-coupled adenosine receptors prevents oxidative DNA damage
Yanling Wua, Jie Zhenga, Joel Lindenb and Joseph Holoshitz ,  , a

a Department of Internal Medicine, University of Michigan, 5520D MSRB1, Ann Arbor, MI 48109-0680, USA
b Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA

Received 12 October 2003;  revised 26 November 2003;  accepted 26 November 2003. ; Available online 24 January 2004.

Abstract
Adenosine has been previously shown to be a cytoprotective paracrine released by injured cells. However, it is presently unknown whether extracellular adenosine can prevent DNA damage. We show here that the adenosine analog, 2-chloroadenosine (2CA), has a potent (IC50=2.04×10-9 M) genoprotective effect in cells subsequently exposed to H2O2. The genoprotective signaling is transduced through adenosine receptors of the A2a and A2b subtypes. Increasing [cAMP]i by forskolin or by cell permeable 8-br-cAMP produced a similar effect, whereas inhibiting the cAMP-mediated pathway with H-89, or increasing [nitric oxide]i or [cGMP]i blocked 2CA effect. Proteasomal inhibitors had no impact on 2CA signaling, while lithium chloride, an inhibitor of glycogen synthase kinase 3 , completely blocked 2CA-induced genoprotective effect. These data indicate for the first time that extracellular adenosine protects cells against imminent oxidative DNA damage and suggest that prophylactic activation of this pathway prior to genotoxic challenges might reduce mutation rates.
Author Keywords: Adenosine; ROS; Genoprotective pathways; Mutation






Daily grape juice consumption reduces oxidative DNA damage and plasma free radical levels in healthy Koreans

Referred to by: Corrigendum to "Daily grape juice consumption reduces oxidative DNA damage and plasma free radical levels in healthy Koreans" [Mutat. Res. 529 (2003) 77–86], Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 546, Issues 1-2, 26 February 2004, Page 103
Yoo Kyoung Parka, Eunju Parkb, Jung-Shin Kima and Myung-Hee Kang ,  , a

Yoo Kyoung Parka, Eunju Parkb, Jung-Shin Kima and Myung-Hee Kang ,  , a
a Department of Food and Nutrition, Hannam University, 306-791, 133 Ojeong-dong, Daedeok-gu, Daejeon 306-791, South Korea
b Division of Life Sciences, Kyungnam University, Masan 631-701, South Korea

Received 10 February 2003;  revised 26 May 2003;  accepted 2 June 2003. ; Available online 22 July 2003.

Abstract
Grape contains flavonoids with antioxidant properties which are believed to be protective against various types of cancer. This antioxidative protection is possibly provided by the effective scavenging of reactive oxygen species (ROS), thus defending cellular DNA from oxidative damage and potential mutations. This study of healthy adults tested whether a daily regimen of grape juice supplementation could reduce cellular DNA damage in peripheral lymphocytes and reduce the amount of free radicals released. Sixty-seven healthy volunteers (16 women and 51 men) aged 19–57 years were given 480 ml of grape juice daily for 8 weeks in addition to their normal diet, and blood samples were drawn before and after the intervention. The DNA damage was determined by using the single cell gel (comet) assay with alkaline electrophoresis and was quantified by measuring tail length (TL). Levels of free radicals were determined by reading the lucigenin-perborate ROS generating source, using the Ultra-Weak Chemiluminescence Analyzer System. Grape juice consumption resulted in a significant decrease in lymphocyte DNA damage expressed by TL (before supplementation: 88.75±1.55  m versus after supplementation: 70.25±1.31  m; P=0.000 by paired t-test). Additionally, grape juice consumption for 8 weeks reduced the ROS/photon count by 15%, compared to the beginning of the study. The preventive effect of grape juice against DNA damage was simultaneously shown in both sexes. These results indicate that the consumption of grape juice may increase plasma antioxidant capacity, resulting in reduced DNA damage in peripheral lymphocytes achieved at least partially by a reduced release of ROS. Our findings support the hypothesis that polyphenolic compounds contained in grape juice exert cancer-protective effects on lymphocytes, limiting oxidative DNA damage possibly via a decrease in free radical levels.
Author Keywords: Grape juice; Antioxidant; Free radical level; DNA damage; Comet assay

Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea

Referred to by: Erratum to "Miscoding properties of 1,N6-ethanoadenine, a DNA adduct derived from reaction with the antitumor agent 1,3-bis(2-chloroethyl)-1-nitrosourea": [Mut. Res. 531 (2003) 191–203], Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, Volume 546, Issues 1-2, 26 February 2004, Page 105
Bo Hang, Ahmed Chenna1, Anton B. Guliaev and B. Singer ,
Abstract | Full Text + Links | PDF (30 K)
Bo Hang, Ahmed Chenna1, Anton B. Guliaev and B. Singer
Life Sciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA

Received 22 February 2003;  revised 26 June 2003;  accepted 10 July 2003. ; Available online 4 November 2003.
Abstract
1,N6-Ethanoadenine (EA) is an exocyclic adduct formed from DNA reaction with the antitumor agent, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). To understand the role of this adduct in the mechanism of mutagenicity or carcinogenicity by BCNU, an oligonucleotide with a site-specific EA was synthesized using phosphoramidite chemistry. We now report the in vitro miscoding properties of EA in translesion DNA synthesis catalyzed by mammalian DNA polymerases (pols)  ,  ,  and  . These data were also compared with those obtained for the structurally related exocyclic adduct, 1,N6-ethenoadenine ( A). Using a primer extension assay, both pols  and  were primarily blocked by EA or  A with very minor extension. Pol  , a member of the Y family of polymerases, was capable of catalyzing a significant amount of bypass across both adducts. Pol  incorporated all four nucleotides opposite EA and  A, but with differential preferences and mainly in an error-prone manner. Human pol  , a paralog of human pol  , was blocked by both adducts with a very small amount of synthesis past  A. It incorporated C and, to a much lesser extent, T, opposite either adduct. In addition, the presence of an A adduct, e.g.  A, could affect the specificity of pol  toward the template T immediately 3' to the adduct. In conclusion, the four polymerases assayed on templates containing an EA or  A showed differential bypass capacity and nucleotide incorporation specificity, with the two adducts not completely identical in influencing these properties. Although there was a measurable extent of error-free nucleotide incorporation, all these polymerases primarily misincorporated opposite EA, indicating that the adduct, similar to  A, is a miscoding lesion.
Author Keywords: BCNU; Ethano adduct; Etheno adduct; Polymerase; Translesion DNA synthesis. Abbreviations: EA, 1,N6-ethanoadenine;  A, 1,N6-ethenoadenine; BCNU, 1,3-bis(2-chloroethyl)-1-nitrosourea; Polymerases  ,  ,  and  , pols  ,  ,  and