Detection of glycosylase, endonuclease and methyltransferase enzyme activities using immobilized oligonucleotides.
A novel rapid assay for detection of DNA glycosylase, restriction endonuclease, and DNA methyltransferase enzyme activities is presented. The assay is based on enzyme-dependent label release (in case of glycosylase and endonuclease), or non-release (in case of methyltransferase) into solution from end-labeled DNA immobilized on solid support (CPG or Tenta Gel S-NH2). The assay has been validated for monitoring activity of repair enzyme uracil-DNA glycosylase, restriction endonucleases SsoII, MvaI and EcoRII and (cytosine-5)-DNA methyltransferase SsoII. Two types of labels have been tested and found compatible with the assay: radioactive (32P) and fluorescent (rhodamine B and fluorescein). The enzyme activity is estimated as a ratio of the label released into solution to the total amount of the label. Use of fluorescent labeling facilitates detection while use of solid phase-immobilized substrates facilitates product separation, improved assay sensitivity, and increases throughput of assay. Proposed technique provides an estimate of enzyme activity but not its specific activity. Thus, the assay will most valuable in the applications where rapid estimation of enzyme activity is necessary.
Sudina
Anna
A
Department of Chemistry, Moscow State University, Moscow, Russia.
Volkov
Eugeny
E
Oretskaya
Tatiana
T
Naryshkin
Nikolai
N
Ivanovskaya
Marina
M
Kubareva
Elena
E
D001345:Autoradiography; D045647:DNA Glycosylases / Q000032:analysis*; D015257:DNA-Cytosine Methylases / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D004720:Endonucleases / Q000032:analysis*; D009841:Oligonucleotides / Q000378:metabolism*
D009841:Oligonucleotides; C063974:DNA modification methylase SsoII; D015257:DNA-Cytosine Methylases; D004720:Endonucleases; D045647:DNA Glycosylases