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Thermostable USER® III Enzyme

Catalog # Concentration Size List Price Quantity Your Price
M5509S 1000 units/ml 50 units $151.00
$135.90
Catalog # Size List Price Your Price
M5509S 50 units $151.00
$135.90
Catalog #
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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.


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    What are endonucleases and their applications?
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. 
 
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  Thermostable USER® III Enzyme M5509SVIAL -20 1 x 0.05 ml 1,000 units/ml
  ThermoPol® Reaction Buffer B9004SVIAL -20 1 x 1.5 ml 10 X
Features
  • Thermostable
  • Leaves 3′-OH and 5′-dRP (different ends than either USER Enzyme or Thermolabile USER II Enzyme)
 
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