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Detection of Glycosylase, Endonuclease and Methyltransferase Enzyme Activities Using Immobilized Oligonucleotides
Author(s) -
Sudina Anna,
Volkov Eugeny,
Oretskaya Tatiana,
Naryshkin Nikolai,
Ivanovskaya Marina,
Kubareva Elena
Publication year - 2004
Publication title -
iubmb life
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.132
H-Index - 113
eISSN - 1521-6551
pISSN - 1521-6543
DOI - 10.1080/15216540410001671925
Subject(s) - dna glycosylase , uracil dna glycosylase , endonuclease , biochemistry , dna , enzyme , dna methyltransferase , oligonucleotide , methyltransferase , chemistry , microbiology and biotechnology , enzyme assay , restriction enzyme , biology , dna repair , methylation
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.IUBMB Life, 56: 139‐143, 2004