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. 2000 Sep 15;28(18):3594-9.
doi: 10.1093/nar/28.18.3594.

Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging

V Rusmintratip  1 , A D Riggs, L C Sowers
Affiliations

Examination of the DNA substrate selectivity of DNA cytosine methyltransferases using mass tagging

V Rusmintratip et al. Nucleic Acids Res. .

Abstract

The biological significance of cytosine methylation is as yet incompletely understood, but substantial and growing evidence strongly suggests that perturbation of methylation patterns, resulting from the infidelity of DNA cytosine methyltransferase, is an important component of the development of human cancer. We have developed a novel in vitro assay that allows us to quantitatively determine the DNA substrate preferences of cytosine methylases. This approach, which we call mass tagging, involves the labeling of target cytosine residues in synthetic DNA duplexes with stable isotopes, such as (15)N. Methylation is then measured by the formation of 5-methylcytosine (5mC) by gas chromatography/mass spectrometry. The DNA substrate selectivity is determined from the mass spectrum of the product 5mC. With the non-symmetrical duplex DNA substrate examined in this study we find that the bacterial methyltransferase HPA:II (duplex DNA recognition sequence CCGG) methylates the one methylatable cytosine of each strand similarly. Introduction of an A-C mispair at the methylation site shifts methylation exclusively to the mispaired cytosine residue. In direct competition assays with HPA:II methylase we observe that the mispaired substrate is methylated more extensively than the fully complementary, normal substrate, although both have one HPA:II methylation site. Through the use of this approach we will be able to learn more about the mechanisms by which methylation patterns can become altered.

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Figures

Figure 1
Figure 1
Sequences of the DNA duplexes used in these assays.
Figure 2
Figure 2
(A) The ion abundance profile for the P–15 peak of silylated unenriched cytosine. (B) The ion abundance profile for the P–15 peak of silylated (4-15NH2)-cytosine. (C) The ion abundance profile for the P–15 peak of the silylated cytosine peak derived from Duplex 1, a 29 bp duplex in which two of 19 cytosine residues are selectively enriched with 15N. The experimentally derived ion abundance at each mass is the solid line to the left of the pair of lines, whereas the dashed line to the right of each pair is the theoretical line calculated.
Figure 3
Figure 3
(A) The ion chromatogram at 270 a.m.u. showing the silylated thymine and 5mC peaks derived from treatment of Duplex 1 with HpaII methylase. (B) The ion abundance profile of the silylated 5mC P–15 peak, derived from treatment of Duplex 1 with HpaII methylase. The experimentally derived ion abundance at each mass is the solid line to the left of the pair of lines, whereas the dashed line to the right of each pair is the theoretical line calculated.
Figure 4
Figure 4
(A) The ion abundance profile of the silylated 5mC P–15 peak, derived from treatment of Duplex 1 with SssI methylase. (B) The ion abundance profile of the silylated 5mC P–15 peak, derived from treatment of Duplex 1 with MspI methylase. The experimentally derived ion abundance at each mass is the solid line to the left of the pair of lines, whereas the dashed line to the right of each pair is the theoretical line calculated.
Figure 5
Figure 5
(A) The ion abundance profile of the silylated 5mC P–15 peak, derived from treatment of Duplex 2 with HpaII methylase. (B) The ion abundance profile of the silylated 5mC P–15 peak, derived from treatment of Duplex 3 with HpaII methylase. (C) The ion abundance profile of the silylated 5mC P–15 peak, derived from treatment of Duplexes 1 and 4 with HpaII methylase. The experimentally derived ion abundance at each mass is the solid line to the left of the pair of lines, whereas the dashed line to the right of each pair is the theoretical line calculated.
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