A thermolabile version is also available, Thermolabile USER II Enzyme (NEB #M5508).
We are excited to announce that all reaction buffers are now BSA-free. NEB began switching our BSA-containing reaction buffers in April 2021 to buffers containing Recombinant Albumin (rAlbumin) for restriction enzymes and some DNA modifying enzymes. Find more details at www.neb.com/BSA-free.
Thermostable USER (Uracil-Specific Excision Reagent) III Enzyme generates a single nucleotide gap at the location of a uracil residue. It is active between 50-75°C (optimal activity observed at 65°C).
Figure 1: Thermostable USER III Enzyme
Afu Uracil DNA glycosylase (UDG) catalyzes the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease IV breaks the phosphodiester backbone at the 3′ and 5′ sides of the abasic site so that base-free deoxyribose is released.
Thermostable USER (Uracil-Specific Excision Reagent) III Enzyme generates a single nucleotide gap at the location of a uracil. Thermostable USER III Enzyme is a mixture of Afu Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease IV. Afu UDG catalyzes the excision of a uracil base, forming an abasic (apyrimidinic) site while leaving the phosphodiester backbone intact. The lyase activity of Endonuclease IV breaks the phosphodiester backbone at the 3′ and 5′ sides of the abasic site so that base-free deoxyribose is released leaving a 5′-deoxyribose phosphate (dRP) and a 3′-hydroxyl. Thermostable USER III Enzyme also has 3′-diesterase activity which can remove 3′ phosphate, 3′-α, β-unsaturated aldehyde, phosphoglycoaldehyde, and other 3' blocking groups. Thermostable USER III Enzyme is active between 50-75°C (optimal activity observed at 65°C).
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.
Source:
The two component proteins are purified separately from E. coli strains containing plasmids encoding Endonuclease IV and Afu UDG.
This product is related to the following categories:
One unit is defined as the amount of enzyme required to nick 10 pmol of a fluorescently labeled 60 mer oligonucleotide duplex containing a single uracil base, in 15 minutes at 65°C in a total reaction volume of 10 µl in 1X Thermopol Buffer.
Reaction Conditions
1X ThermoPol® Reaction Buffer
Incubate at 65°C
1X ThermoPol® Reaction Buffer
20 mM Tris-HCl
10 mM (NH4)2SO4
10 mM KCl
2 mM MgSO4
0.1% Triton® X-100
(pH 8.8 @ 25°C)
Storage Buffer
15 mM Tris-HCl
35 mM NaCl
1 mM DTT
0.1 mM
100 µg/ml BSA
50% Glycerol
pH 7.4 @ 25°C
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