rE. coli MutY with Buffer

E. coli MutY DNA Glycosylase acts together with E. coli Formamidopyrimidine-DNA Glycosylase, or Fpg, to prevent the potentially mutagenic consequences of 8-oxo-dG lesions. 8-oxo-dG lesions escaping repair by Fpg frequently pair with Adenine (A) during DNA replication, producing an 8-oxo-dG:A mispair. MutY removes the A from this base pair to initiate base excision repair. In the absence of MutY, DNA replication after an 8-oxo-dG:A mismatch results in thymine incorporation opposite the adenine in one of the daughter strands, creating a fixed mutation. MutY has an associated AP lyase activity.

4 pmoles of A/G mismatch oligonucleotide set with the A oligo end-labeled, 1X REC Buffer 4 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 10 mM EDTA), and serial dilutions of enzyme in a 20 μL reaction volume are incubated for 1 hour at 37 °C. To complete cleavage of a basic site, fresh NaOH is added to final concentration of 166 mM then heated for 15 minutes at 95 °C. For analysis, 24 μL of 2X Loading Buffer (20 mM EDTA, 97% formamide, and 0.2% bromophenol blue) is added, the samples are heated at 95 °C for 5 min then fast cooled to 2-8 °C, and the cleavage products are resolved by 20% denaturing polyacrylamide gel electrophoresis. The bands are analyzed to quantify the cleavage products.