Product Class: Other

Thermostable USER® III Enzyme
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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.

 

Product Introduction

Mechanism of Thermostable USER® III

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).
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Catalog # Size Concentration
M5509S 50 units 1000 units/ml

Product Information

Description

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 1: Thermostable USER III Enzyme

Workflow showing mechanism of Thermostable USER® III versus Afu UDG and Endonuclease IV

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.


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

Comparison of mechanisms of USER Enzyme, USER II Enzyme and USER III Enzyme

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:
DNA Repair Enzymes and Structure-specific Endonucleases
This product can be used in the following applications:
DNA Assembly and Cloning

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Unit Definition

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

Features

  • Thermostable
  • Leaves 3′-OH and 5′-dRP (different ends than either USER Enzyme or Thermolabile USER II Enzyme)
 

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is the activity of Thermostable USER® III Enzyme in other buffers?
  2. What temperature is Thermostable USER® III Enzyme active?
  3. How is Thermostable USER® III Enzyme different than USER Enzyme and Thermolabile USER II Enzyme?
  4. Will Thermostable USER® III Enzyme remove uracil from ssDNA and dsDNA?
  5. What is the difference between the suite of USER® enzymes (#M5505, #M5508, #M5509) and Thermostable Endonuclease Q (#M0701)?
  6. Do any of the USER enzymes leave ligatable ends when cleaving at a Uracil?
  7. How close to the ends of DNA can Thermostable USER III Enzyme (NEB #M5509) cleave?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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