Characterizing DNA methyltransferases with an ultrasensitive luciferase-linked continuous assay

Ivan Hemeon, Jemy A. Gutierrez, Meng Chiao Ho, Vern L. Schramm

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

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′-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 (0.1-1000 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 kcat but not Km for AdoMet. The assay is shown to be robust in the presence of high concentrations of the pyrimidine analogues 5-azacytidine and 5-azacytosine.

Original languageEnglish (US)
Pages (from-to)4996-5004
Number of pages9
JournalAnalytical Chemistry
Volume83
Issue number12
DOIs
StatePublished - Jun 15 2011

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Methyltransferases
Luciferases
Assays
DNA
Adenine
Adenosine Monophosphate
Methionine
adenosylhomocysteine nucleosidase
Orthophosphate Dikinase Pyruvate
DNA (Cytosine-5-)-Methyltransferase
Adenosine Triphosphate
Firefly Luciferases
Azacitidine
Kinetics
Cytosine
Homocysteine
Transcription
Transferases
Labeling
Recycling

ASJC Scopus subject areas

  • Analytical Chemistry

Cite this

Characterizing DNA methyltransferases with an ultrasensitive luciferase-linked continuous assay. / Hemeon, Ivan; Gutierrez, Jemy A.; Ho, Meng Chiao; Schramm, Vern L.

In: Analytical Chemistry, Vol. 83, No. 12, 15.06.2011, p. 4996-5004.

Research output: Contribution to journalArticle

Hemeon, Ivan ; Gutierrez, Jemy A. ; Ho, Meng Chiao ; Schramm, Vern L. / Characterizing DNA methyltransferases with an ultrasensitive luciferase-linked continuous assay. In: Analytical Chemistry. 2011 ; Vol. 83, No. 12. pp. 4996-5004.
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