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

Mullins EA, Rubinson EH, Pereira KN, Calcutt MW, Christov PP, Eichman BF
Methods. 2013 64 (1): 59-66

PMID: 23876937 · PMCID: PMC3812247 · DOI:10.1016/j.ymeth.2013.07.020

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 (3 mA) and N7-methylguanine (7 mG) 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.

Copyright © 2013 Elsevier Inc. All rights reserved.

MeSH Terms (12)

Alkylation Bacillus cereus Bacterial Proteins Chromatography, High Pressure Liquid DNA DNA Adducts DNA Damage DNA Repair Humans Saccharomyces cerevisiae Proteins Salmonella typhi Tandem Mass Spectrometry

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