PAGE separation of hemi-methylated or unmethylated oligonucleotide substrates to distinguish between maintenance and de novo DNA methyltranferase activity.
DNA methyltransferase (DNMT) enzymes catalyze the addition of a methyl group to cytosine residues in DNA. Appropriate cytosine methylation of CpG dinucleotides is required for normal mammalian development and homeostasis, and quantitative methods are necessary to assess DNMT activity in various cell extracts. The method described in this report utilizes incorporation of S-[methyl-(3)H]-adenosyl-L-methionine into hemi-methylated or unmethylated oligonucleotides to distinguish between maintenance and de novo DNMT activity, respectively. However, unlike previously described methods, this protocol uses native polyacrylamide gel electrophoresis to detect the incorporation of radioactivity into substrate oligonucleotides. This approach distinguishes between incorporation of radioactivity into target substrate oligonucleotides and incorporation into non-specific cellular DNA that often contaminates nuclear extracts, and permits the reproducible quantitation and comparison of de novo and maintenance DNMT activities in various cell lines. Electrophoretic separation of the methylated substrates is a cost-effective, specific, and reproducible approach to quantitate DNMT activities in nuclear extracts.
Butler
Jill S
JS
Herman B Wells Center of Pediatric Research, Section of Pediatric Hematology/Oncology, Departments of Pediatrics and Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202, United States.
Lee
Jeong-Heon
JH
Skalnik
David G
DG
D002457:Cell Extracts / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000032:analysis / Q000378:metabolism*; D019175:DNA Methylation; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005821:Genetic Techniques; D007553:Isotope Labeling; D009841:Oligonucleotides / Q000302:isolation & purification* / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D002457:Cell Extracts; D009841:Oligonucleotides; D012436:S-Adenosylmethionine; D004248:DNA (Cytosine-5-)-Methyltransferases