Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase

Abstract

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.

Keywords: DNA damage; DNA repair; cytosine; deamination; glycosylase; mass spectrometry; mutation.

Figure 1

Pathway for mutations induced by deamination of cytosine and analogs. Deaminated intermediates can be converted to mutation by DNA replication or repair synthesis. Mispaired intermediates can also be repaired by excision repair pathways.

Figure 2

Amino acid sequence of hyTDG. The 29 amino acid peptide derived from hTDG is shown in red and functionally significant peptides observed by mass spectrometry are shown in bold. hTDG, human thymine DNA glycosylase; hyTDG, hybrid thymine DNA glycosylase.

Figure 3

Mass spectrum of peptide KVDR/LDDATNK, which forms the junction between hTDG and MIG sequences. The sequence of the peptide is determined from examination of the b or y ion fragments as indicated in the figure. hTDG, human thymine DNA glycosylase; MIG, thymine DNA glycosylase from Methanobacterium thermoautotrophicum.

Figure 4

Comparison of UDG, hyTDG, and hTDG activity using a gel cleavage assay. Oligonucleotides with a 5′-FAM label (2.5 pmol) containing U or T at the target site were either unpaired or paired with complementary sequences to form U:A or T:A base pairs or U:G or T:G mispairs. Oligonucleotides were incubated for 1 h with UDG (2.5 units, 0.04 μg, 1.6 pmol) at 37 °C, hyTDG (0.5 μg, 16.8 pmol) at 65 °C, or hTDG (1.5 μg, 31.0 pmol) at 37 °C. Sodium hydroxide was then added to hydrolyze the phosphate backbone of oligonucleotides containing an abasic site, and oligonucleotides were resolved by PAGE, visualized, and quantified with a STORM imager. UDG cleaves all uracil-containing oligonucleotides completely but has no activity on T. The hyTDG cleaves only mispaired U (95%) and T (86%). The hTDG cleaves U mispaired with G (73%) but much less T mispaired with G (6%). 6-FAM, 6-carboxyfluorescein; hTDG, human thymine DNA glycosylase; hyTDG, hybrid thymine DNA glycosylase; UDG, uracil-DNA glycosylase.

Figure 5

Analysis of glycosylase activity of hyTDG on oligonucleotides using a real-time fluorescence assay. In the absence (A) and presence (B) of added calf thymus DNA. About 25 pmol duplexes with 5′-FAM (upper strand) and 3′-BHQ1 (lower strand) were incubated with 25 pmol hyTDG at 65 °C in the presence of DMDA, which cleaves abasic sites. Fluorescence was monitored in a Roche 480 qPCR instrument. The equation for the solid lines in each figure is Y = A(1 − e^−kt), where Y is the percent oligonucleotide cleaved, A is maximum percent cleaved, k is the rate constant (min⁻¹), and t is time in minutes. In panel A, the values of A and k for U:G are 96.2 ± 0.6% and 0.1075 ± 0.103 min⁻¹ and for T:G are 92.4 ± 1.10% and 0.0694 ± 0.009 min⁻¹. In panel B, the experiment is identical to panel A, except that 20 μg of calf thymus DNA was added. Values of A and k in the presence of calf thymus for U:G are 99.9 ± 1.1% and 0.1136 ± 0.0147 min⁻¹ and for T:G 103 ± 1.1% and 0.0613 ± 0.0122 min⁻¹. The rate of U:G cleavage exceeds T:G, by factors of 1.6 to 1.9. The rate of U:G and T:G cleavage does not change significantly upon addition of calf thymus DNA. BHQ1, black hole fluorescence quencher 1; DMDA, N,N-dimethylethylenediamine; FAM, 6-carboxyfluorescein; hyTDG, hybrid thymine DNA glycosylase; qPCR, quantitative PCR.

Figure 6

Workflow for measuring bases released by hyTDG or UDG using mass spectrometry. Oligonucleotides or DNA are incubated with hyTDG or UDG in the presence of one or more stable-isotope standards. Following incubation, free bases are isolated by spin filtration. Isolated bases are derivatized and analyzed by GC–EI–MS/MS or GC–NCI–MS. Pyrimidines released by the glycosylase are quantified by comparing the integrated peak area of the unenriched pyrimidine with the peak area of a corresponding stable isotope–enriched standard. Created in part with BioRender.com. EI, electron ionization; hyTDG, hybrid thymine DNA glycosylase; NCI, negative chemical ionization; UDG, uracil-DNA glycosylase.

Figure 7

Cleavage of a mixture of oligonucleotides containing T:G and U:G mispairs by hyTDG followed simultaneously by gel electrophoresis and GC–MS/MS. Mixtures of 5′-FAM-labeled oligonucleotides containing U:G (8.3 pmol) or T:G (16.7 pmol) were incubated with 250 pmol hyTDG and isotope-enriched standards (U + 3, T + 4) in a total volume of 25 μl at 65 °C. At selected time intervals, 5 μl was used for gel electrophoresis, and the remaining 20 μl was used for the measurement of released bases. Released bases were separated by spin filtration, derivatized, and analyzed by GC–MS/MS. Gel analysis indicated predominant cleavage of U:G and T:G oligonucleotides by hyTDG by 60 min (panel A). The oligonucleotide mixture was also incubated with UDG at 37 °C (1 unit, 0.6 pmol), which cleaved the U:G but not T:G oligonucleotide (panel A, far right). Base release was measured by GC–MS/MS as shown in panel B. Each time point was analyzed three times. At 2 h, 6.42 ± 0.49 pmol U and 13.33 ± 0.34 pmol were released, representing nearly complete release of U:G and T:G in the sample. The amount of U released by UDG at 2 h was 7.58 ± 0.2 pmol. FAM, 6-carboxyfluorescein; hyTDG, hybrid thymine DNA glycosylase.

Figure 8

Analysis of the release of U and mispaired T from calf thymus DNA by UDG and hyTDG. Approximately 400 μg of EcoRI-digested calf thymus DNA was incubated with UDG (10 units, 6.2 pmol, 37 °C) or hyTDG (295 pmol, 65 °C) for 90 min. Released bases were isolated by spin filtration, derivatized, and analyzed by GC–EI–MS/MS (panel A) or GC–NCI–MS (panel B). Data presented above represents observed amounts minus background from three independent experiments. In panel A, total uracil (single stranded, U:A and U:G) released by UDG was 9.39 ± 0.29 pg/μg DNA. The amount of uracil from U:G released by hyTDG was 1.30 ± 0.29 pg/μg, and the amount of T from T:G released was 5.58 ± 0.42 pg/μg. These amounts correspond to one deaminated U:G mispair per 4.48 × 10⁴ C:G base pairs and one deaminated T:G mispair per 6.71 × 10² 5-mC:G base pairs. In panel B, total uracil (single strand, U:A and U:G) released by UDG was 8.46 ± 0.63 pg/μg DNA. The amount of uracil from U:G released by hyTDG was 0.54 ± 0.13 pg/μg, and the amount of T from T:G released was 4.14 ± 0.21 pg/μg. These amounts correspond to one deaminated U:G mispair per 1.08 × 10⁵ C:G base pairs and one deaminated T:G mispair per 9.09 × 10² 5-mC:G base pairs. Most of the U is in U:A base pairs or single-stranded DNA (86% panel A, 94% panel B). The amount of T in T:G mispairs exceeds the amount of U in U:G mispairs by a factor of 4.3 (panel A) to 7.7 (panel B). EI, electron ionization; hyTDG, hybrid thymine DNA glycosylase; NCI, negative chemical ionization; UDG, uracil-DNA glycosylase.