Effects of 6-Thioguanine and S6-Methylthioguanine on Transcription in Vitro and in Human Cells

You, Changjun; Dai, Xiaoxia; Yuan, Bi-Feng; Wang, Yinsheng

doi:10.1074/jbc.m112.418681

Cited by 22 publications

(39 citation statements)

References 53 publications

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“…129–135 As illustrated in Fig. 7, a double-stranded plasmid harboring a site-specifically incorporated and structurally defined lesion is allowed to replicate in cultured human cells.…”

Section: Qualitative Analysis Of Dna Adductsmentioning

confidence: 99%

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

2015

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Exogenous and endogenous sources of chemical species can react, directly or after metabolic activation, with DNA to yield DNA adducts. If not repaired, DNA adducts may compromise cellular functions by blocking DNA replication and/or inducing mutations. Unambiguous identification of the structures and accurate measurements of the levels of DNA adducts in cellular and tissue DNA constitute the first and important step towards understanding the biological consequences of these adducts. The advances in mass spectrometry (MS) instrumentation in the past 2–3 decades have rendered MS an important tool for structure elucidation, quantification, and revelation of the biological consequences of DNA adducts. In this review, we summarized the development of MS techniques on these fronts for DNA adduct analysis. We placed our emphasis of discussion on sample preparation, the combination of MS with gas chromatography-or liquid chromatography (LC)-based separation techniques for the quantitative measurement of DNA adducts, and the use of LC-MS along with molecular biology tools for understanding the human health consequences of DNA adducts. The applications of mass spectrometry-based DNA adduct analysis for predicting the therapeutic outcome of anti-cancer agents, for monitoring the human exposure to endogenous and environmental genotoxic agents, and for DNA repair studies were also discussed.

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“…129–135 As illustrated in Fig. 7, a double-stranded plasmid harboring a site-specifically incorporated and structurally defined lesion is allowed to replicate in cultured human cells.…”

Section: Qualitative Analysis Of Dna Adductsmentioning

confidence: 99%

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

2015

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“…The mixture was loaded onto a 30% polyacrylamide gel (acrylamide:bis-acrylamide ϭ 19:1) and products were quantified by phosphorimager analysis. Similar to a previously described method for assessing the impact of DNA lesions on transcription (22,44), we determined the effects of DNA lesions on replication efficiency and fidelity by the "relative bypass efficiency" (RBE) and "relative mutation frequency" (RMF) values, respectively. Briefly, the RMF value was determined from the relative amounts of different products arising from replication of the lesion-containing genome.…”

Section: Pcr Page and Lc-ms/ms Analyses-mentioning

confidence: 99%

“…The RBE value was calculated using the following formula, %RBE ϭ (lesion signal/competitor signal)/(non-lesion control signal/ competitor signal) (45,46). LC-MS/MS was used to further identify the replication products arising from S-cdA-or S-cdG-bearing substrates similar to those described elsewhere (22,29,37,44). Briefly, PCR products were treated with 50 units of NcoI and 20 units of shrimp alkaline phosphatase in 250 l of New England Biolabs buffer 3 at 37°C for 4 h, followed by heating at 80°C for 20 min.…”

Section: Pcr Page and Lc-ms/ms Analyses-mentioning

confidence: 99%

Translesion Synthesis of 8,5′-Cyclopurine-2′-deoxynucleosides by DNA Polymerases η, ι, and ζ

You¹,

Swanson

Dai³

et al. 2013

Journal of Biological Chemistry

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145

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Background:The 8,5Ј-cyclopurine-2Ј-deoxynucleosides (cPus) are important types of oxidative DNA damage. Results: cPus exhibit both inhibitory and mutagenic effects on replication; polymerases (Pols) , , and are involved in translesion synthesis of these lesions. Conclusion: Pols , , and cooperatively promote translesion synthesis across cPu lesions. Significance: This work revealed the effects of cPus on DNA replication in mammalian cells.

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“…This is followed by PAGE and LC-MS/MS analyses of short DNA fragments released from the restriction digestion of RT-PCR products 8 . This method was inspired by the lesion bypass and mutagenesis assays that have been successfully used for determining the effects of DNA lesions on replication in Escherichia coli cells 9–12 , and it has been used for determining the effects of a variety of DNA lesions on transcription in vitro and in mammalian cells 8,13–16 .…”

Section: Introductionmentioning

confidence: 99%

Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo

You

Wang

2015

Nat Protoc

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Aberrant transcription induced by DNA damage may confer risk for the development of cancer and other human diseases. Traditional methods for measuring lesion-induced transcriptional alterations often involve extensive colony screening and DNA sequencing procedures. Here we describe a protocol for the quantitative assessment of the effects of DNA lesions on the efficiency and fidelity of transcription in vitro and in mammalian cells. The method is also amenable to investigating the influence of specific DNA repair proteins on the biological response toward DNA damage during transcription by manipulating their gene expression. Specifically, we present detailed, step-by-step procedures, including DNA template preparation, in vitro and in vivo transcription, RNA purification, reverse-transcription PCR (RT-PCR) and restriction digestion of RT-PCR products. Analyses of restriction fragments of interest are performed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) and polyacrylamide gel electrophoresis (PAGE). The entire procedure described in this protocol can be completed in 15–20 d.

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Effects of 6-Thioguanine and S6-Methylthioguanine on Transcription in Vitro and in Human Cells

Cited by 22 publications

References 53 publications

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

Mass spectrometry for the assessment of the occurrence and biological consequences of DNA adducts

Translesion Synthesis of 8,5′-Cyclopurine-2′-deoxynucleosides by DNA Polymerases η, ι, and ζ

Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo

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