Characterizing DNA Methyltransferases With An Ultrasensitive Luciferase-Linked Continuous Assay

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• 作者：Ivan Hemeon ; Jemy A. Gutierrez ; Meng-Chiao Ho ; Vern L. Schramm
• 刊名：Analytical Chemistry
• 出版年：2011
• 出版时间：June 15, 2011
• 年：2011
• 卷：83
• 期：12
• 页码：4996-5004
• 全文大小：917K
• 年卷期：v.83,no.12(June 15, 2011)
• ISSN：1520-6882

文摘

DNA (cytosine-5)-methyltransferases (DNMTs) catalyze the transfer of a methyl group from S-adenosyl-l-methionine (AdoMet) to the 5-position of cytosine residues and thereby silence transcription of regulated genes. DNMTs are important epigenetic targets. However, isolated DNMTs are weak catalysts and are difficult to assay. We report an ultrasensitive luciferase-linked continuous assay that converts the S-adenosyl-l-homocysteine product of DNA methylation to a quantifiable luminescent signal. Results with this assay are compared with the commonly used DNA labeling from [methyl-³H]AdoMet. A 5鈥?methylthioadenosine-adenosylhomocysteine nucleosidase is used to hydrolyze AdoHcy to adenine. Adenine phosphoribosyl transferase converts adenine to AMP and pyruvate orthophosphate dikinase converts AMP to ATP. Firefly luciferase gives a stable luminescent signal that results from continuous AMP recycling to ATP. This assay exhibits a broad dynamic range (0.1鈥?000 pmol of AdoHcy). The rapid response time permits continuous assays of DNA methylation detected by light output. The assay is suitable for high-throughput screening of chemical libraries for DNMT inhibition activity. The kinetic properties of human and bacterial CpG methyltransferases are characterized using this assay. Human catalytic domain DNMT3b activation by DNMT3L is shown to involve two distinct kinetic states that alter k_cat but not K_m for AdoMet. The assay is shown to be robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine.