Home FAQs How is Thermostable USER® III Enzyme different than USER Enzyme and Thermolabile USER II Enzyme?

FAQ: How is Thermostable USER® III Enzyme different than USER Enzyme and Thermolabile USER II Enzyme?

Thermostable USER III Enzyme is similar to USER (Uracil-Specific Excision Reagent) Enzyme (NEB #M5505) and Thermolabile USER II Enzyme (NEB #M5508), in that all 3 will generate a single nucleotide gap at the location of a uracil. Thermolabile USER II Enzyme has the added benefit of being heat-inactivatable (65°C for 10 minutes), while Thermostable USER III Enzyme is active at higher temperatures (>50°C). Thermolabile USER II can be heat inactivated, while USER Enzyme and Thermostable USER III Enzyme must be removed from the reaction via other means (column cleanup or other purification methods). All USER enzymes will break the phosphodiester backbone but leave different 3′ and 5′ ends: the lyase activity of Endonuclease VIII in USER Enzyme leaves a 3′ phosphate and 5′ phosphate, the lyase activity of Endonuclease III in Thermolabile USER II Enzyme leaves a 3′-phospho-α, β-unsaturated aldehyde and 5′ phosphate and the lyase activity of Endonuclease IV in Thermostable USER III Enzyme leaves a 3′-hydroxyl and 5′-deoxyribose phosphate.

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.