UNG E.Coli Active
Greater than 97.0% as determined by SDS-PAGE.
Description
UNG E.Coli Recombinant produced in E.Coli is a single, non-glycosylated polypeptide UNG is purified by proprietary chromatographic techniques.
Product Specs
Uracil DNA glycosylase (UDG), also known as uracil-DNA glycosylase 1, is an essential enzyme present in all life forms. Its primary function is to repair damaged DNA by selectively removing uracil bases. These uracil bases can be erroneously incorporated into DNA during replication or arise from cytosine deamination. UDG is known by various names depending on the organism, including b2580, JW2564, EC 3.2.2.27, DGU, UNG15, HIGM5, EC 3.2.2, HIGM4, and UNG2. This description focuses on E. coli UDG, exploring its structure, function, and significance in molecular biology.
Structurally, E. coli UDG belongs to the uracil DNA glycosylase (UDG) superfamily, as revealed by extensive studies on its crystal structure. The E. coli UDG monomer consists of 229 amino acids, resulting in a molecular weight of 25 kDa. Its structure is predominantly composed of beta-sheets, with an alpha-helix positioned on one side and a DNA-binding groove on the other. The active site of E. coli UDG houses a conserved glutamic acid residue. This residue acts as a catalytic base, facilitating the hydrolysis of the N-glycosidic bond between uracil and the sugar-phosphate backbone of DNA.
Functionally, E. coli UDG plays a critical role in safeguarding the integrity of the genome. It achieves this by preventing the accumulation of mutations that can arise from the presence of uracil in DNA. Uracil can appear in DNA through two main pathways: spontaneous deamination of cytosine or incorporation during DNA synthesis when dUTP is utilized instead of dTTP. If left unrepaired, uracil bases can lead to DNA damage and genomic instability, potentially resulting in cell death or disease. E. coli UDG specifically targets and removes these uracil bases from DNA, creating an abasic site. This site is then further processed and repaired by other dedicated repair enzymes.
Recombinant UNG from E. coli is produced in E. coli. This single, non-glycosylated polypeptide UNG undergoes purification using proprietary chromatographic methods.
The UNG protein solution (5U/ul) contains the following components: 10mM Tris-HCl (pH 7.4 at 25°C), 50mM KCl, 0.1 mM EDTA, 1mM DTT, 0.1mg/ml BSA, and 50% glycerol.
Adding a carrier protein (0.1% HSA or BSA) is recommended for long-term storage.
Avoid repeated freeze-thaw cycles.
One unit of enzyme activity is defined as the amount of UNG that catalyzes the release of 60 picomoles of uracil per minute from double-stranded DNA containing uracil. The activity is measured at 37°C for 30 minutes in a 50 microliter reaction mixture containing 0.2 micrograms of DNA (with a specific activity of 104-105 counts per minute per microgram).
The purity of UNG is determined by SDS-PAGE to be greater than 97.0%.
Treating 0.1 micrograms of uracil-containing DNA with 1 unit of UDG for 10 minutes at 37°C renders the DNA unsuitable for copying by DNA polymerase. The enzyme can be 95% heat-inactivated by incubation at 95°C for 10 minutes. However, because UDG retains some activity even after heat treatment at 95°C, it is recommended to add a uracil glycosylase inhibitor to prevent the degradation of the product DNA. Alternatively, phenol/chloroform extraction can be performed immediately after the reaction to isolate the reaction products.
Product Science Overview
Recombinant UDG is typically produced using Escherichia coli (E. coli) as a host organism. The gene encoding UDG is cloned into an expression vector, which is then introduced into E. coli cells. These cells are cultured under conditions that promote the expression of the UDG enzyme. The recombinant enzyme is subsequently purified from the bacterial cells for use in various applications .
UDG catalyzes the hydrolysis of the N-glycosidic bond between the uracil base and the sugar-phosphate backbone of DNA. This reaction releases free uracil and creates an abasic site (AP site) in the DNA. The AP site is then processed by other enzymes in the base excision repair pathway to restore the correct DNA sequence .
Recombinant UDG has several important applications in molecular biology and biotechnology:
- PCR Contamination Prevention: UDG is used to prevent carryover contamination in PCR reactions. By incorporating uracil into PCR products and treating them with UDG, any contaminating DNA from previous reactions can be degraded, ensuring the specificity of the PCR .
- DNA Repair Studies: UDG is employed in research to study DNA repair mechanisms and the role of uracil in DNA damage and mutagenesis .
- Site-Directed Mutagenesis: UDG can be used to create specific mutations in DNA by excising uracil residues and allowing for targeted modifications .
