New England Biolabs, M0464S, Thermostable OGG

Related Categories DNA Repair Enzymes and Structure-specific Endonucleases Specification Unit Definition One unit is defined as the amount of enzyme required to cleave 10 pmol of a 60-mer fluorescently labeled oligonucleotide duplex containing a single 8-oxoguanine base paired with a cytosine in a total reaction volume of 10 μl in 1 hour at 65°C. Reaction Conditions 1X rCutSmart™ Buffer Incubate at 65°C 1X rCutSmart™ Buffer 50 mM Potassium Acetate 20 mM Tris-acetate 10 mM Magnesium Acetate 100 µg/ml Recombinant Albumin (pH 7.9 @ 25°C) Storage Buffer 10 mM Tris-HCl 500 mM NaCl 0.1 mM EDTA 1 mM DTT 200 µg/ml Recombinant Albumin 50% Glycerol pH 7.4 @ 25°C FAQ Q: What is the difference between Thermostable OGG (NEB #M0464) and Fpg (NEB #M0240)? A: Both Fpg and Thermostable OGG are bifunctional glycosylases, meaning the glycosylase activity first recognizes and removes the modified base in DNA, then an AP-lyase activity cleaves the DNA backbone. Thermostable OGG, unlike FPG, is thermostable (active at temperatures as high as 80°C) and while the two enzymes have similar substrate specificity (both recognize and cut at 8-oxoguanine), they cleave the DNA backbone differently. Thermostable OGG cuts the bond 3′ to an 8-oxoguanine base. This leaves a nick in the DNA substrate with a 5´ phosphate and a 3´-phospho-α, β-unsaturated aldehyde. Fpg cuts both 5′ and 3′′ to the modified base. The modified base floats away creating a 1 nucleotide DNA gap with 5´ and 3´ phosphate termini. Q: What is the activity of Thermostable OGG in other buffers? A: Thermostable OGG has activity in the following buffers: NEBuffer r1.1=100% activity NEBuffer r2.1=75% activity NEBuffer r3.1=50% activity rCutSmart Buffer=100% activity Thermopol Buffer=100% activity Q: Will Thermostable OGG cut an 8-oxoguanine paired with a base other than cytosine? A: While Thermostable OGG preferentially cleaves at the modified base (8-oxoG) when it is base paired with cytosine (C), the enzyme will cleave at the 8-oxoG regardless of what base is paired with the 8-oxoguanine. Q: What is the molecular weight of Thermostable OGG? A: The molecular weight of Thermostable OGG is 31,376 Da. Q: Is Thermostable OGG a tagged protein? A: Yes, Thermostable OGG contains a C-terminal hexa-histidine tag. Q: What substrate is used to test Thermostable OGG? A: The substrates used to test enzyme activity are listed under the unit definition for each enzyme. Thermostable OGG is tested using a fluorescently labeled 60-mer oligonucleotide duplex containing a single 8-oxoguanine base paired with cytosine. Q: Does Thermostable OGG cut only one strand, or both, to cause a double-strand break? A: Thermostable OGG cuts only the strand that contains the damage. If there is damage on both strands, then it will cut both. Q: What modified bases will Thermostable OGG recognize and cleave? A: Thermostable OGG specifically recognizes and cuts 8-oxoguanine. Thermostable OGG will not cut other modified bases including 8-oxoadenine, 5-hydroxycytosine, 5-hyrdroxyuracil or thymine glycol. Q: Will Thermostable OGG cut 8-oxoguanine in ssDNA? A: Yes, Thermostable OGG will cleave ssDNA containing an 8-oxoguanine, however the activity on ssDNA is significantly reduced (~32-fold) as compared to activity on a dsDNA substrate. Q: What temperatures is Thermostable OGG active? A: Thermostable OGG is active between 25-80°C, however optimal activity is observed between 65-75°C. Temperature (°C) Units/ml 16 1000 25 1000 37 2500 42 4500 50 5500 60 7000 65 8000 70 8000 75 8000 80 6000 Activity at different temperatures is based on the unit definition assay: The amount of enzyme required to cleave 10 pmol of a 60-mer fluorescently labeled oligonucleotide duplex containing a single 8-oxoguanine base paired with cytosine in a total reaction volume of 10 μl in 1 hour.