Direct Analysis of Enzyme-Catalyzed DNA Demethylation

被引:16
|
作者
Karkhanina, Anna A. [1 ]
Mecinovic, Jasmin [2 ,3 ]
Musheev, Michael U. [1 ]
Krylova, Svetlana M. [1 ]
Petrov, Alexander P. [1 ]
Hewitson, Kirsty S. [2 ,3 ]
Flashman, Emily [2 ,3 ]
Schofield, Christopher J. [2 ,3 ]
Krylov, Sergey N. [1 ]
机构
[1] York Univ, Dept Chem, Toronto, ON M3J 1P3, Canada
[2] Univ Oxford, Dept Chem, Oxford OX1 3TA, England
[3] Univ Oxford, Oxford Ctr Integrat Syst Biol, Oxford OX1 3TA, England
基金
加拿大自然科学与工程研究理事会; 英国惠康基金; 英国生物技术与生命科学研究理事会;
关键词
HUMAN ALKB PROTEINS; ESCHERICHIA-COLI; OXIDATIVE DEMETHYLATION; FTO GENE; DAMAGE; REPAIR; FLUORESCENCE; SUBSTRATE; BACTERIAL; OBESITY;
D O I
10.1021/ac9010556
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
N/O-methylation of DNA can be cytotoxic and mutagenic; therefore, enzymes that reverse DNA methylation are essential for organism survival. Several 2-oxoglutarate-dependent oxygenases and methyltransferases that remove a methyl group from a methylated DNA base have been identified. Studies of their kinetics and search for their inhibitors have been retarded by the lack of an approach to directly quantitate DNA substrates and products that differ by a single methyl group. Here, we introduce such an approach, which is based on capillary electrophoresis with laser-induced fluorescence detection. We achieved baseline separation of a fluorescently labeled 15-nucleotide-long single-base methylated DNA substrate from its demethylated product, followed by its quantitative detection. We then used this approach to study the kinetics of AlkB-catalyzed DNA demethylation and screen a number of potential inhibitors of this reaction. Ten new inhibitors, which can be used as templates in developing therapies targeting AlkB-like enzymes, were identified. Our approach will be applicable for in vitro kinetic studies of known DNA demethylating and methylating enzymes and in the discovery of new ones.
引用
收藏
页码:5871 / 5875
页数:5
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