DNA Damage and Cytotoxicity Induced by Minor Groove Binding Methyl Sulfonate Esters

Varadarajan, S.; Shah, Deesha; Dande, Prasad; Settles, Samuel; Chen, Fa Xian; Fronza, Gilberto; Gold, Barry

doi:10.1021/bi0353272

Cited by 22 publications

(25 citation statements)

References 41 publications

(92 reference statements)

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“…Of interest is 3mA, a product of endogenous methyl donors (e.g., S-adenosylmethionine) [67, 68] and alkylating agents used in chemotherapy [69, 70]. Due to the short half-life of 3mA in DNA [71], it has not been possible to prepare purified oligonucleotides containing a single 3mA modification suitable for use in biochemical assays, although methylsulfonate derivatized lexitropsin (Me-Lex) peptides have been useful to study the biological effects of 3mA [72, 73]. The data presented here represents the first kinetic characterization of 3mA release by AlkD, and reveals that AlkD excises 3mA at a rate two orders of magnitude greater than 7mG, suggesting that 3mA may be the preferred substrate of AlkD.…”

Section: Discussionmentioning

confidence: 99%

An HPLC–tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products

Mullins

Rubinson

Pereira

et al. 2013

Methods

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DNA glycosylases excise a broad spectrum of alkylated, oxidized, and deaminated nucleobases from DNA as the initial step in base excision repair. Substrate specificity and base excision activity are typically characterized by monitoring the release of modified nucleobases either from a genomic DNA substrate that has been treated with a modifying agent or from a synthetic oligonucleotide containing a defined lesion of interest. Detection of nucleobases from genomic DNA has traditionally involved HPLC separation and scintillation detection of radiolabeled nucleobases, which in the case of alkylation adducts can be laborious and costly. Here, we describe a mass spectrometry method to simultaneously detect and quantify multiple alkylpurine adducts released from genomic DNA that has been treated with N-methyl-N-nitrosourea (MNU). We illustrate the utility of this method by monitoring the excision of N3-methyladenine (3mA) and N7-methylguanine (7mG) by a panel of previously characterized prokaryotic and eukaryotic alkylpurine DNA glycosylases, enabling a comparison of substrate specificity and enzyme activity by various methods. Detailed protocols for these methods, along with preparation of genomic and oligonucleotide alkyl-DNA substrates, are also described.

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Section: Discussionmentioning

confidence: 99%

An HPLC–tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products

Mullins

Rubinson

Pereira

et al. 2013

Methods

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“…In addition, the methylation selectivity at N3-A is greater than 90% so that the 3-mA (minor groove) to 7-mG (major groove) ratio exceeds 100 : 1 as compared to the 1 : 10 ratio observed with DMS and MMS [30]. There is a strong correlation between 3-mA formation and cytotoxicity [31, 32]. Moreover, the cytotoxicity is strongly enhanced in cells lacking the 3-methyadenine glycosylase [16, 25, 33] or by treatment of repair proficient cells with antisense oligomers targeted against the glycosylase [34].…”

Section: Discussionmentioning

confidence: 99%

Effect of N3-Methyladenine and an Isosteric Stable Analogue on DNA Polymerization

Settles

Wang

Fronza³

et al. 2010

Journal of Nucleic Acids

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N3-methyladenine (3-mA) is a cytotoxic lesion formed by the reaction of DNA with many methylating agents, including antineoplastic drugs, environmental agents and endogenously generated compounds. The toxicity of 3-mA has been attributed to its ability to block DNA polymerization. Using Me-lex, a compound that selectively and efficiently reacts with DNA to afford 3-mA, we have observed in yeast a mutational hotspot at the 5′-terminus of an A4 tract. In order to explore the potential role of sequence-dependent DNA polymerase bypass of 3-mA, we developed an in vitro system to prepare 3-mA modified substrates using Me-lex. We detail the effects of 3-mA, its stable isostere analogue, 3-methyl-3-deazaadenine, 3-deazaadenine and an THF abasic site on DNA polymerization within an A4 sequence. The methyl group on 3-mA and 3-methyl-3-deazaadenine has a pronounced inhibitory effect on DNA polymerization. There was no sequence selectivity for the bypass of any of the lesions, except for the abasic site, which was most efficiently by-passed when it was on the 5′-terminus of the A4 tract. The results indicate that the weak mutational pattern induced by Me-lex may result form the depurination of 3-mA to an abasic site that is bypassed in a sequence dependent context.

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“…Although there was considerable cytotoxicity, likely dependent from the preferential induction (90-95 %) of the lethal 3-MeA adduct, the highest mutation frequency observed after high Me-lex concentrations was only 3 fold over the background. This result is in contrast with the mutation induction of MMS, a methylating agent inducing the mutagenic 7-MeG and the 3-MeA adducts in a 10:1 ratio [25]. Indeed, after treatment with a comparable cytotoxic concentration of MMS, the mutation frequency at the Hprt locus in the same CHO cell line was about 40 fold the background [23].…”

Section: Discussionmentioning

confidence: 99%

High frequency of genomic deletions induced by Me-lex, a sequence selective N3-adenine methylating agent, at the Hprt locus in Chinese hamster ovary cells

Russo¹,

Fronza²,

Ottaggio³

et al. 2009

Mutation Research/Fundamental and Molecular Mechanisms of Mutag

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We have investigated the mutagenicity induced at the Hprt locus in Chinese hamster ovary (CHO) cells treated with increasing concentrations of Me-lex, a minor groove selective methylating agent that efficiently generates more than 90-95% of 3-MeA DNA adducts. Me-lex treatment was cytotoxic but weakly mutagenic, resulting in up to 7-fold induction above background in the Hprt mutation frequency. The molecular nature of 43 Hprt mutations induced by Me-lex was determined by sequence analysis of the Hprt cDNA and genomic analysis of the gene locus. Base pair substitutions represented about 25% of Me-lex induced mutations. The mutation spectrum revealed a high percentage of genomic deletions (51%) comprising single/multiple exon(s) and even the loss of the complete locus. When the distribution of mutations among different classes was considered, the difference between the spontaneous and Me-lex induced CHO spectra was statistically significant (p<0.012), indicating that the sites where mutations occurred were Me-lex specific. Based upon these results we hypothesize that a large proportion of mutations may result from the processing of 3-MeA, the main adduct induced by Me-lex, within A/T rich sequences in non-coding regions of the Hprt gene. The processing of these lesions by DNA polymerases could result in recombination and genomic deletions, thus representing a severe threat for genome integrity.

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DNA Damage and Cytotoxicity Induced by Minor Groove Binding Methyl Sulfonate Esters

Cited by 22 publications

References 41 publications

An HPLC–tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products

An HPLC–tandem mass spectrometry method for simultaneous detection of alkylated base excision repair products

Effect of N3-Methyladenine and an Isosteric Stable Analogue on DNA Polymerization

High frequency of genomic deletions induced by Me-lex, a sequence selective N3-adenine methylating agent, at the Hprt locus in Chinese hamster ovary cells

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