A column method for determination of DNA cytosine-C5-methyltransferase activity.
DNA methylation at the 5th position of cytosine has been found to be correlated with tumorigenesis. An inhibitor of DNA methylase could, therefore, be used as an anticancer drug. However, only a few inhibitory compounds have been discovered due to the limitations for assaying the DNA methylation. In this study, we describe a modification of DNA cytosine-C5-methyltransferase assay system utilizing [(3)H]-labeled S-adenosyl-methionine (SAM) and Sephadex G-25 column. Pre-treatment of either lambda DNA or the promoter region of human telomerase (hTERT) with HaeIII methylase greatly reduced the digestion of the DNAs with the corresponding restriction enzyme HaeIII endonuclease (over 100-fold), and the result was further confirmed by agarose gel electrophoresis. Application of this column method to another modification/restriction system, EcoRI methylase/endonuclease, gave rise to the similar results. Our data suggest that the newly developed column method could be effective for rapid screening of large number of cytosine methylase inhibitors and could also be applicable to other DNA methylases.
Kim
Bo Yeon
BY
Laboratory of Cellular Signaling Modulator, Korea Research Institute of Bioscience and Biotechnology, 305-333 Yuseong, Daejon, South Korea.
Kwon
Oh Sung
OS
Joo
Sung Ah
SA
Park
Jung Ah
JA
Heo
Keon Young
KY
Kim
Min Soo
MS
Ahn
Jong Seog
JS
D010582:Bacteriophage lambda / Q000235:genetics; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000378:metabolism; D003911:Dextrans; D006801:Humans; D011401:Promoter Regions, Genetic / Q000235:genetics; D019098:Telomerase / Q000235:genetics
D004269:DNA, Bacterial; D003911:Dextrans; C025614:sephadex; D004248:DNA (Cytosine-5-)-Methyltransferases; D019098:Telomerase