Uracil DNA glycosylase (UDG), or uracil-DNA glycosylase 1, is a crucial enzyme found in all life forms, involved in repairing damaged DNA by specifically removing uracil bases that are misincorporated into DNA during replication or deaminated cytosine. In various organisms, UDG goes by different names, such as b2580, JW2564, EC 22.214.171.124, DGU, UNG15, HIGM5, EC 3.2.2, HIGM4, and UNG2. Here, we delve into the E. coli UDG, examining its structure, function, and applications in molecular biology.
Structure: The crystal structure of E. coli UDG has been extensively studied, revealing that it belongs to the uracil DNA glycosylase (UDG) superfamily. The E. coli UDG monomer has 229 amino acids with a molecular weight of 25 kDa. The protein has a beta-sheet-rich structure with an alpha-helix on one side and a groove on the other side that binds to DNA. The active site of E. coli UDG contains a conserved glutamic acid residue that acts as a catalytic base to facilitate the hydrolysis of the N-glycosidic bond between uracil and the sugar phosphate backbone.
Function: E. coli UDG plays a critical role in maintaining the integrity of the genome by preventing the accumulation of mutations that can arise from the incorporation of uracil into DNA. Uracil in DNA can occur spontaneously from the deamination of cytosine or can be incorporated during DNA synthesis when dUTP is used instead of dTTP. Unrepaired uracil bases can lead to DNA damage and genomic instability, possibly resulting in cell death or disease. E. coli UDG specifically recognizes and removes uracil bases from DNA, creating an abasic site that is further processed by other repair enzymes.
UNG E.Coli Recombinant produced in E.Coli is a single, non-glycosylated polypeptide UNG is purified by proprietary chromatographic techniques.
UNG protein solution (5U/ul) containing 10mM Tris-HCl (25℃, pH 7.4), 50mM KCl, 0.1 mM EDTA, 1mM DTT, 0.1mg/ml BSA & 50% glycerol.
For long term storage it is recommended to add a carrier protein (0.1% HSA or BSA).
Avoid multiple freeze-thaw cycles.
1 unit is defined as the amount of enzyme that catalyzes the release of 60pmol of uracil/minute from double-stranded, uracil-containing DNA. Activity is measured by release of [3H]-uracil in a 50µl reaction containing 0.2µg DNA (104-105 cpm/µg) in 30 min. at 37°C.
Greater than 97.0% as determined by SDS-PAGE.
Treatment of 0.1μg of uracil containing DNA with 1U UDG for 10 min. at 37℃ renders the DNA incapable of being copied by DNA polymerase. The enzyme can be 95% heat killed by incubation at 95℃ for 10 minutes. Since UDG remains partially active following heat treatment at 95℃, it is recommended that uracil glycosylase inhibitor be added to prevent degradation of product DNA. Alternatively, reaction products can be immediately extracted with phenol/chloroform