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A nonradioactive restriction enzyme-mediated assay to detect DNA repair by Fe(II)/2-oxoglutarate-dependent dioxygenase

Authors
Publisher
Elsevier Inc.
Publication Date
Identifiers
DOI: 10.1016/j.ab.2014.07.003
Keywords
  • Fe(Ii)/2-Oxoglutarate-Dependent Dioxygenase
  • Alkb
  • Methyl Methanesulfonate
  • Dna Repair
Disciplines
  • Biology

Abstract

Abstract The Escherichia coli DNA repair enzyme AlkB belongs to the Fe(II)/2-oxoglutarate-dependent dioxygenase family. It removes methyl groups from 1-methyl adenine (1-meA) and 3-methyl cytosine (3-meC) lesions present in single-stranded DNA by oxidative decarboxylation. In the current article, we describe an in vitro assay that permits rapid detection of AlkB activity. To achieve this, we generated methylated oligonucleotide using methyl methanesulfonate and then monitored DNA repair using a methylation-sensitive restriction enzyme and novel agarose gel electrophoresis system capable of resolving small oligonucleotides. Our approach overcomes several drawbacks of NAD+-dependent formaldehyde dehydrogenase-coupled assay and radioisotope-based assay for determining AlkB DNA repair activity.

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