Endonuclease III (Nth)

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).

Highlights

  • Isolated from a recombinant source
  • Supplied with 10X Reaction Buffer

Product Source

An E. coli strain which carries the cloned nth gene from Escherichia coli.

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
Endonuclease III (Nth) Reaction Buffer-2010X

Advantages and Features

Applications

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

Properties and 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 Temperature

-20°C

Storage Conditions

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 min

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

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.

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 CutSmart buffer?

Protocols

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

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Selection Charts

Usage Guidelines & Tips

Interactive Tools

Citations

  • Karbaschi, Mahsa., Macip, Salvador., Mistry, Vilas., Abbas, Hussein H.K., Delinassios, George J., Evans, Mark D., Young, Antony R., Cooke, Marcus S. (2015). Rescue of cells from apoptosis increases DNA repair in UVB exposed cells: implications for the DNA damage response Toxicol. Res.. 4, 725-738. DOI: doiL 10.1039/c4tx00197d

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page. Further information regarding NEB product quality can be found here.
  • Endonuclease Activity (Nicking):
    The product is tested in a reaction containing a supercoiled DNA substrate. After incubation for 4 hours the percent converted to the nicked form is determined by agarose gel electrophoresis.
  • Exonuclease Activity (Radioactivity Release):
    The product is tested in a reaction containing a radiolabeled mixture of single and double-stranded DNA. After incubation for 4 hours the exonuclease activity is determined by the % release of radioactive nucleotides.
  • Non-Specific DNase Activity (16 hour):
    The product is tested for non-specific nuclease degradation in a reaction containing a DNA substrate. After incubation for 16 hours there is no detectable degradation of the DNA substrate as determined by agarose gel electrophoresis.

Safety Data Sheet

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

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.