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

Abstract

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.

Keywords: 1,N(6)-ethenoadenine; 1mA; 3mA; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; 6-carboxyfluorescein; 7mG; AAG; AlkA; Alkylation; BSA; Base excision repair; CID; DMS; DNA glycosylase; DTT; E. coli 3-methyladenine DNA glycosylase I; E. coli 3-methyladenine DNA glycosylase II; EDTA; ESI; ESI(+); FAM; HEPES; HPLC; MAG; MMS; MNNG; MNU; MRM; MS/MS; Mass spectrometry; Methylpurine; N-methyl-N-nitrosourea; N-methyl-N′-nitro-N-nitrosoguanidine; N1-methyladenine; N3-methyladenine; N7-methylguanine; TAG; Tris; bovine serum albumin; collision induced dissociation; dimethylsulfate; dithiothreitol; electrospray ionization; ethylenediaminetetraacetic acid; high performance liquid chromatrography; human alkyladenine DNA glycosylase; methyladenine DNA glycosylase; methylmethanesulfonate; multiple reaction monitoring; positive ion mode electrospray ionization; tandem mass spectrometry; tris(hydroxymethyl)aminomethane; εA.

Figure 1

Alkylpurine DNA glycosylase reaction and substrates. (A) Schematic of the DNA glycosylase reaction. (B) Structures of the major methylated purines produced by laboratory alkylating agents. Percentages refer to the relative amounts of methylpurines produced by MNU treatment [2].

Figure 2

Two methods for monitoring DNA glycosylase base excision activity. (A) Oligonucleotide assay. A ³²P- or fluorescently labeled (star) oligonucleotide containing a modified nucleobase (asterisk) is incubated with a DNA glycosylase to generate an AP site. At various time points, aliquots of the reaction are mixed with NaOH and heated to nick any AP sites. The substrate (S) and product (P) bands are separated by denaturing polyacrylamide gel electrophoresis, shown schematically to the right. (B) Genomic DNA assay. Genomic DNA is treated with a methylating agent (e.g., N-methyl-N-nitrosourea, MNU) to produce a spectrum of methylated nucleobases (gray ovals), some of which are excised from the DNA backbone by DNA glycosylases. The glycosylase reaction is quenched and spiked with deuterated internal standards (dark gray ovals before the genomic DNA is ethanol precipitated and the soluble fraction containing free methylbases is subjected to HPLC/MS-MS. Ions were generated in positive ion electrospray ionization [ESI(+)] mode. MS/MS detection was based on multiple reaction monitoring of highly selective collision induced dissociation (CID) transitions with a triple quadrupole mass spectrometer.

Figure 3

Characterization of base excision from genomic DNA using HPLC-MS/MS. (A) Comparison of 3mA (black bars) and 7mG (gray bars) excision activities of several alkylpurine DNA glycosylases. Methylated genomic DNA was incubated with either 5 N HCl or 5 μM glycosylase for 1 h at 37°C. Enzymes used: human Δ79AAG, S. cerevisiae MAG, S. typhi TAG wild-type and E38A mutant, B. cereus AlkC, and B. cereus AlkD wild-type and D113N mutant. The numbers used to generate this graph are provided in Supplemental Table S2. (B) Enzyme concentration dependence of 3mA (black circles) and 7mG (gray squares) excision by AlkD. Reactions containing 0.01-5 μM AlkD were incubated at 37°C for 1 h. (C) Time dependence of 3mA (black circles) and 7mG (gray squares) excision by AlkD. Reactions containing 0.5 μM AlkD were incubated at 37°C for 1 min to 10 h. Reaction mixtures were equilibrated at 37°C for 3 min prior to the addition of enzyme.

Figure 4

Excision of 7mG from an oligonucleotide substrate by human Δ79AAG. Reactions contained 5 μM Δ79AAG, 100 nM FAM-DNA, 50 mM sodium acetate (pH 6.0), 100 mM NaCl, 1 mM DTT, 1 mM EDTA, and 0.1 mg/ml BSA. (A) Denaturing polyacrylamide gel showing 25mer 7mG-DNA (S) and AP-nicked 13mer product (P) at various reaction times. (B) Quantitation of the data in panel A.