Product Class: Other

Endonuclease III (Nth)
NEBU cloned at NEB recombinant 37 65 Heat

Product Introduction

Diagram of mechanism

  • Bifunctional DNA glycosylase with DNA N-glycosylase and AP lyase activities
  • The N-glycosylase activity releases damaged pyrimidines, including thymine glycol and 5, 6-dihydroxythymine, generating an AP site. The AP lyase activity cleaves an AP site via β-elimination, creating a 1-nucleotide gap with 3'-α, β-unsaturated aldehyde and 5'-phosphate termini
Catalog # Size Concentration
M0268S 1000.0 units 10000 units/ml

Product Information

Description

Endonuclease III (Nth) protein from E. coli acts as both N-glycosylase and a AP-lyase. The N-glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating a basic (AP site). The AP-lyase activity of the enzyme cleaves 3´ to the AP site leaving a 5´ phosphate and a 3´ ring opened sugar. Some of the damaged bases recognized and removed by Endouclease III include urea, 5, 6 dihydroxythymine, thymine glycol, 5-hydroxy-5 methylhydanton, uracil glycol, 6-hydroxy-5, 6-dihdrothimine and methyltartronylurea (1,2).

Product Source

An E. coli strain which carries the cloned nth gene from Escherichia coli.
This product is related to the following categories:

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • M0268S     -20    
      Endonuclease III (Nth) M0268SVIAL -20 1 x 0.1 ml 10,000 units/ml
      Endonuclease III (Nth) Reaction Buffer B0268SVIAL -20 1 x 1 ml 10 X

Properties & Usage

Unit Definition

One unit is defined as the amount of enzyme required to cleave 1 pmol of a 34 mer oligonucleotide duplex containing a single AP site* in a total reaction volume of 10 µl in 1 hour at 37°C in 1X Endonuclease III Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplex.

* An AP site is created by treating 10 pmol of a 34 mer oligonucleotide duplex containing a single uracil residue with 1 unit of Uracil-DNA Glycosylase (UDG) for 2 minutes at 37°C.

Reaction Conditions

1X Endonuclease III (Nth) Reaction Buffer
Incubate at 37°C

1X Endonuclease III (Nth) Reaction Buffer
20 mM Tris-HCl
1 mM EDTA
1 mM DTT
(pH 8 @ 25°C)

Usage Concentration

10,000 units/ml

Storage Buffer

10 mM Tris-HCl
250 mM NaCl
1 mM DTT
0.1 mM EDTA
200 µg/ml BSA
50% Glycerol
pH 7.4

Heat Inactivation

65°C for 20 minutes

Unit Assay Conditions

1X Endonuclease III (Nth) Reaction Buffer containing 10 pmol of fluorescently labeled oligonucleotide duplexes in a total reaction volume of 10 µl

Application Features

  • Single cell gel electrophoresis (Comet assay) (3,4,5)
  • Alkaline elution (6)
  • Alkaline unwinding (7)

Product Notes

  1. Effective with M0268S/L spec V2, NEB has updated the product specification as follows:
  2. Protein Purity Assay (SDS-PAGE):  Endonuclease III (Nth) is ≥95% pure as determined by SDS PAGE analysis using Coomassie Blue detection.
  3. qPCR DNA Contamination (E. coli Genomic):  A minimum of 1 μl of Endonuclease III (Nth) is screened for the presence of E. coli genomic DNA using SYBR® Green qPCR with primers specific for the E. coli 16S rRNA locus.  Results are quantified using a standard curve generated from purified E. coli genomic DNA. The measured level of E. coli genomic DNA contamination is ≤1 E. coli genome.
  4. Endonuclease Activity (Nicking): Removed to align with standard quality control testing for product portfolio.

References

  1. Dizdaroglu, M., Laval, J. and Boiteux, S. (1993). Biochemistry. 32, 12105-12111.
  2. Hatahet, Z. et al. (1994). J. Biol. Chem.. 269, 18814-18820.
  3. Singh, N. et al. (1988). Exp. Cell Res.. 175, 184-191.
  4. Collins, A. et al. (1993). Carcinogenesis. 14, 1733-1735.
  5. Collins, A. et al. (1996). Environ. Health Perspect . 104, 465-469.
  6. Pflaum, M. et al. (1998). Free Rad. Res.. 29, 585-594.
  7. Hartwig, A. et al. (1996). Toxicol. Lett. 88, 85-90.

Protocols, Manuals & Usage

Protocols

  1. Comet Assay - Modified for Detection of Oxidized Bases Using the Repair Endonucleases Fpg, hOGG1 and Endonuclease III (Nth)

Usage & Guidelines

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What is the activity of Endonuclease III in the NEBuffer 1-4?
  2. What is the molecular weight of Endonuclease III?
  3. What types of damaged bases are recognized and removed by Endonuclease III?
  4. Is Endonuclease III a tagged protein?
  5. What substrate is used to test Endonuclease III activity?
  6. Will Endonuclease III work in rCutSmart buffer?

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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