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DNA Modifying Enzymes and Cloning >
DNA Repair Proteins >
Endonuclease V |
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Prices are in US dollars and valid only for US orders.
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- Isolated from a recombinant source
- Supplied with 10X Reaction Buffer
Description:
Endonuclease V is a repair enzyme found in E. coli that recognizes deoxyinosine, a deamination product of deoxyadenosine in DNA. Endonuclease V, often called deoxyinosine 3´ endonuclease, recognizes DNA containing deoxyinosines (paired or not) on double-stranded DNA, single-stranded DNA with deoxyinosines and to a lesser degree, DNA containing abasic sites (ap) or urea, base mismatches, insertion/deletion mismatches, hairpin or unpaired loops, flaps and pseudo-Y structures. It is believed that Endonuclease V needs another protein to repair the DNA, as it does not remove the deoxyinsoine or the damaged bases (1,2,3).
Endonuclease V cleaves the second and third phosphodiester bonds 3´ to the mismatch of deoxyinosine with a 95% efficiency for the second bond and a 5% efficiency for the third bond (2), leaving a nick with 3´-hydroxyl and 5´-phosphate (4).
Source:
An E. coli strain containing a gene fusion of the Endo V gene and the gene coding for the maltose binding protein (MBP). The fusion protein is purified to near homogeneity and is active as a fusion. The protein contains 223 amino acids and has a molecular weight of 24.9 kDa (5).
Applications:- Mismatch Cleavage
- Cleavage of oligonucleotides containing deoxyinosine and a weaker affinity for oligonucleotides containing base mismatches (5)
Reagents Supplied:
NEBuffer 4 (10X)
Enzyme Properties
Heat Inactivation:
No
Reaction & Storage Conditions
Reaction Conditions:
1X NEBuffer 4
Incubate at
37°C.
1X NEBuffer 4:
20 mM Tris-acetate
50 mM potassium acetate
10 mM Magnesium Acetate
1 mM Dithiothreitol
pH 7.9 @ 25°C
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 deoxyinosine site* in a total reaction volume of 10 µl in 1 hour at 37°C.
* A deoxyinosine site is synthetically prepared with a dl in the middle of one strand of a 34 mer oligonucleotide duplex.
Concentration:
10,000 units/ml
Storage Conditions:
10 mM Tris-HCl
300 mM NaCl
1 mM Dithiothreitol
1 mM EDTA
50% Glycerol
0.15% Triton X-100
pH 8.0 @ 25°C
Storage Temperature:
-20°C
Notes
Usage notes:- When using a buffer other than the optimal (supplied) NEBuffer, it may be necessary to add more enzyme to achieve complete digestion.
Quality Control for Current Lot
Quality control values for a specific lot can be found on the datacard which accompanies each product.
Exonuclease Activity:
Incubation of a 50 μl reaction containing 10 units of Endonuclease V with 1 μg of a mixture of single and double-stranded
[3H] E. coli DNA (205 cpm/μg) for 4 hours at 37ºC
released < 0.1% of the total radioactivity.
References
- Yao, M. and Kow, Y.W. (1996) J. Biol. Chem., 271, 30672-30673.
- Yao, M. and Kow, Y.W. (1995) J. Biol. Chem., 270, 28609-28616.
- Yao, M. et. al. (1994) J. Biol. Chem., 269, 31390-31396.
- He, B., Qing, H. and Kow, Y.W. (2000) Mutat. Res., 459, 109-114.
- Yao, M. and Kow, Y.W. (1994) J. Biol. Chem. , 269, 31390-31396.
Reagents Sold Separately
NEBuffer 4
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