A bioluminescence assay for DNA methyltransferase activity based on methylation-resistant cleavage.
DNA methyltransferase (MTase) is a kind of important regulatory factor in various biological processes. Current methods to investigate DNA MTase activity are still limited in the sensitivity and/or generality. Therefore, developing methods with high sensitivity and improved generality is needed. Here, we develop a new bioluminescence strategy based on methylation-resistant cleavage and protein expression in vitro to detect DNA MTase activity. In the strategy, Dam MTase was used as a model enzyme and MboI as the methylation-resistant endonuclease, and luciferase reporter DNA (LR-DNA) was used as their action target. Because the completely methylated LR-DNA could be expressed as detectable luciferase, Dam MTase activity was quantified by measuring the luminescence intensity of the expressed luciferase. The assay provides a very low detection limit (0.08 U/ml) as well as a wide linear range (0.2-100 U/ml). Besides, the analysis mode has improved generality and could be extended to the detection of other DNA MTases and the corresponding inhibitor screening.
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China.
Cheng
C
Jiang
Chun-Yan
CY
Yan
Cheng
C
Huang
Jian-Hui
JH
Jiang
Ru-Qin
RQ
Yu
D004247:DNA / Q000378:metabolism*; D053837:DNA Cleavage; D019175:DNA Methylation; D003850:Deoxyribonuclease I / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D017930:Genes, Reporter; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*
D004247:DNA; D008156:Luciferases; C058693:Dam methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D003850:Deoxyribonuclease I