Site-specific mutagenesis: retrospective and prospective

Singer, Brett C.; Essigmann, John M.

doi:10.1093/carcin/12.6.949

Cited by 97 publications

(7 citation statements)

References 34 publications

(45 reference statements)

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“…1 Alkylation at nitrogen atoms on nucleobases has been shown to induce transversions, frameshift mutations, and small deletions. 2 Alkylation at oxygen atoms, albeit occurring to a lesser extent than N -alkylation, 3,4 primarily produces point mutations and is linked with the carcinogenic and mutagenic properties of many alkylating agents. 5 For larger alkylating agents, the reactivity of oxygen atoms in nucleobases differs, with the O 6 position of guanine being the most reactive, followed by the O 2 and O 4 positions of thymine, and then the O 2 position of cytosine.…”

Section: Introductionmentioning

confidence: 99%

In-Vitro Replication Studies on O²-Methylthymidine and O⁴-Methylthymidine

Andersen

Wang

et al. 2012

Chem. Res. Toxicol.

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O2- and O4-methylthymidine (O2-MdT and O4-MdT) can be induced in tissues of laboratory animals exposed with N-methyl-N-nitrosourea, a known carcinogen. These two O-methylated DNA adducts have been shown to be poorly repaired and may contribute to the mutations arising from exposure to DNA methylating agents. Here, in vitro replication studies with duplex DNA substrates containing site-specifically incorporated O2-MdT and O4-MdT showed that both lesions blocked DNA synthesis mediated by three different DNA polymerases, including the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (Kf−), human DNA polymerase κ (pol κ) and Saccharomyces cerevisiae DNA polymerase η (pol η). Results from steady-state kinetic measurements and LC-MS/MS analysis of primer extension products revealed that Kf− and pol η preferentially incorporated the correct nucleotide (dAMP) opposite O2-MdT, while O4-MdT primarily directed dGMP misincorporation. While steady-state kinetic experiments showed that pol κ-mediated nucleotide insertion opposite O2-MdT and O4-MdT is highly promiscuous, LC-MS/MS analysis of primer extension products demonstrated that pol κ incorporated favorably the incorrect dGMP opposite both lesions. Our results underscored the limitation of the steady-state kinetic assay in determining how DNA lesions compromise DNA replication in vitro. In addition, the results from our study revealed that, if left unrepaired, O-methylated thymidine lesions may constitute important sources of nucleobase substitutions emanating from exposure to alkylating agents.

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Section: Introductionmentioning

confidence: 99%

In-Vitro Replication Studies on O²-Methylthymidine and O⁴-Methylthymidine

Andersen

Wang

et al. 2012

Chem. Res. Toxicol.

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“…10,11 Quantitation of ethylated DNA adducts in human tissues could provide an approach to investigate the possible role of direct ethylating agents in human cancer. With this goal in mind, we previously developed a liquid chromatography-electrospray ionization-tandem mass spectrometry-selected reaction monitoring method for quantitation of 7-ethyl-Gua in human hepatic DNA.…”

Section: Introductionmentioning

confidence: 99%

Quantitation of 7-Ethylguanine in Leukocyte DNA from Smokers and Nonsmokers by Liquid Chromatography–Nanoelectrospray-High Resolution Tandem Mass Spectrometry

Balbo

Villalta

Hecht

2011

Chem. Res. Toxicol.

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There is considerable evidence for exposure of humans to an unknown ethylating agent, and some studies indicate that cigarette smoking may be one source of this exposure. Therefore, we have developed a liquid chromatography-nanoelectrospray-high resolution tandem mass spectrometry-selected reaction monitoring (LC-NSI-HRMS/MS-SRM) method for the analysis of 7-ethyl-Gua in human leukocyte DNA, a readily available source of DNA. [15N5]7-Ethyl-Gua was used as the internal standard. Leukocyte DNA was isolated and treated by thermal neutral hydrolysis. The hydrolysate was partially purified by solid-phase extraction. The fraction containing 7-ethyl-Gua was analyzed by LC-NSI-HRMS/MS-SRM using the transition m/z 180 [M + H]+ → m/z 152.05669 [Gua + H]+ for 7-ethyl-Gua and m/z 185 → m/z 157.04187 for the internal standard. The detection limit was approximately 10 amol on column, while the limit of quantitation was about 8 fmol/μmol Gua starting with 180 μg DNA (corresponding to 36 μg DNA on-column). Leukocyte DNA samples from 30 smokers and 30 nonsmokers were analyzed. Clear peaks for 7-ethyl-Gua and the internal standard were observed in most of the samples. The mean (± S.D.) level of 7-ethyl-Gua measured in leukocyte DNA from smokers was 49.6 ± 43.3 (range 14.6 – 181) fmol/μmol Gua while that from nonsmokers was 41.3 ± 34.9 (range 9.64 – 157) fmol/μmol Gua. Although a significant difference between smokers and nonsmokers was not observed, the method described here is unique in the use of high resolution mass spectrometry and establishes for the first time the presence of 7-ethyl-Gua in human leukocyte DNA.

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“…However, persistent miscoding of adducts compromises DNA repair systems resulting in accumulation of mutations in the genome. Multiple studies have indicated that cigarette smoke derived DNA adducts are responsible for miscoding mutations (9)(10)(11). Mutations in tumor suppressor genes or oncogenes enables uncontrolled growth and aid in development of cancer (12).…”

Section: Introductionmentioning

confidence: 99%