USER™ Enzyme |
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USER (Uracil-Specific Excision
Reagent) Enzyme (1) generates a single nucleotide
gap at the location of a uracil. USER Enzyme is a
mixture of Uracil DNA glycosylase (UDG) and the
DNA glycosylase-lyase Endonuclease VIII. UDG
catalyses the excision of a uracil base, forming an
abasic (apyrimidinic) site while leaving the phosphodiester
backbone intact (2,3). The lyase activity
of Endonuclease VIII breaks the phosphodiester
backbone at the 3´ and 5´ sides of the abasic site
so that base-free deoxyribose is released (4,5). To help select the best DNA assembly method for your needs, please use our Synthetic Biology/DNA Assembly Selection Chart. Figure 2: USER Enzyme (NEB #M5505), Thermolabile USER II Enzyme (NEB #M5508) and Thermostable USER III Enzyme (NEB #M5509) generate different functional ends after cleavage of DNA.
In addition to different optimal reaction temperatures (37°C for USER and Thermolabile USER II Enzyme and 65°C for Thermostable USER III Enzyme) and ability to be heat inactivated (Thermolabile USER II Enzyme only), the different USER Enzymes generate different 3’ and 5’ termini after cleavage. USER Enzyme (NEB #M5505) contains Endonuclease VIII and leaves a 3’ and 5’ phosphate after cleavage. Thermolabile USER II Enzyme (NEB #M5508) contains Endonuclease III and leaves a 3′-phospho-α, β-unsaturated aldehyde and 5′ phosphate after cleavage. Thermostable USER III Enzyme (NEB #M5509) contains Endonuclease IV and leaves a 3′-hydroxyl and 5′-deoxyribose phosphate.
Product Source The two component proteins are purified separately from E. coli K-12 strains containing plasmids encoding Endonuclease VIII and Uracil-DNA Glycosylase. This product is related to the following categories: DNA Repair Enzymes and Structure-specific Endonucleases Products This product can be used in the following applications: Applications of USER® and Thermolabile USER II Enzymes, USER® Cloning, DNA Assembly and Cloning, High-throughput cloning and automation solutions |
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