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Radiation Damage and Alkylation Damage Enzymes
E. coli Endonuclease IV
E. coli Exonuclease III
E. coli Uracil-N-Glycosylase
Mouse
3-Methyladenine DNA Glycosylase Type II (Aag Protein)
| Endonuclease IV is a
class II AP endonuclease with an associated 3-diesterase activity and
no associated N-glycosylase activity. Endonuclease IV can remove
phosphoglycoaldehyde, deoxyribose-5-phosphate, 4-hydroxy-2-pentanal, and
phosphate groups from the 3 ends of DNA. Endonuclease IV does not
contain 3 exonuclease activity. |
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| Purified from E. coli containing a
recombinant plasmid harboring the E. coli nfo gene. |
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| One unit is the amount of enzyme required
to cleave an AP-site oligonucleotide within an oligonucleotide duplex at
the rate of 1 pmol/hour at 37°C. |
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| 1X REC
Buffer 1 (10 mM HEPES-KOH, pH 7.4, 100 mM KCl), 4 pmoles AP-site
oligonucleotide (Cat# 3851-100-01) labeled with 32P, 4 pmoles
complementary oligonucleotide (Cat# 3851-100-02), and 0.01-1 Unit
(serially diluted) endonuclease IV in a 20 ΅l reaction volume and
incubate for 1 hour at 37°C. Cleavage products are resolved by 20%
denaturing PAGE. |
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| Endonuclease IV exhibits AP endonuclease
and 3- repair diesterase activities. |
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CometAssay/FLARE
 |
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Supercoiled DNA
relaxation assay
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Alkaline elution assay |
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| Freeze in working aliquots at -80°C to
avoid repeated freeze-thawing. The enzyme is supplied in 10 mM HEPES-KOH
(pH 7.4), 100 mM KCl, 0.1 mg/ml BSA, and 50% glycerol. |
Products
|
4050-100-EB |
E. coli Endonuclease IV, 100 Units and 10X REC Buffer 1, 1 ml |
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4050-500-EB |
E. coli Endonuclease IV, 500 Units and 10X REC Buffer 1, 1 ml |
| Exonuclease III is an AP
endonuclease with associated 3-5 exonuclease activity that acts on 3
OH, 3 phosphate, and 3 phosphoglycolate groups. Exonuclease III may
also act upon 3 α, β unsaturated aldehydes generated following β
elimination at AP sites. |
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| Purified from E. coli containing a
recombinant plasmid harboring the E. coli xth A gene. |
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| One unit is the amount of enzyme required
to produce 1 nmole of acid-soluble total nucleotide in 30 minutes at
37°C. |
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| 1X REC
Buffer 1 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl), 0.5 pmol
AP-oligonucleotide (Cat# 3851-100-01) labeled with 32P, 0.5
pmole complementary oligonucleotide (Cat#3851-100-02), and 5 units of
Exonuclease III in a 20 ΅l reaction volume. Following incubation for 1
hour at 37°C, cleavage products are resolved by 20% denaturing
polyacrylamide gel electrophoresis. |
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E. coli
exonuclease III cleaves 5 to AP sites. It also has 3 diesterase, 3-
to 5- exonuclease, and RNase H activities. The enzyme is stimulated by
Mg2+ and requires double-stranded
substrates. |
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CometAssay/FLARE
|
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Supercoiled DNA
relaxation assay
|
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Alkaline elution assay |
|
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| Freeze at -80°C in working aliquots to
avoid repeated freeze-thaw. The enzyme is supplied in a buffer
containing 50% glycerol. |
Products
|
4030-05K-EB |
E. coli
Exonuclease III, 5000 Units and 10X REC Buffer 1, 1 ml |
| Uracil bases in DNA can
arise from deamination of cytosine giving rise to increased spontaneous
mutations. The enzyme Uracil-N-Glycosylase removes uracil from the DNA
leaving an AP site.
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| Purified from E. coli containing a
recombinant plasmid harboring the E. coli ung gene. |
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| The amount of enzyme required to cleave a
uracil-containing oligonucleotide within an oligonucleotide duplex at
the rate of 60 pmole/min. at 37°C. |
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| 1 X REC
Buffer 6 (20 mM Tris-Cl (pH 8.0), 1mM EDTA, 1 mM DTT, 0.1 mg/ml BSA), 4
pmoles of uracil oligonucleotide (Cat# 3852-100-01) labeled with 32P,
2 pmoles of oligo complement B (Cat# 3849-100-02), and
Uracil-N-Glycosylase in a 20 ΅l reaction volume. The tubes are incubated
for 1 hour at 37°C and then treated with 0.1 M NaOH for 15 minutes at
95°C. The cleavage products are resolved by 20% denaturing PAGE. |
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| Uracil-N-Glycosylase releases uracil,
5-hydroxy-2-deoxyuridine, 5,6-dihydroxyuracil, 5-fluorouracil from both
double or single stranded DNA. |
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CometAssay/FLARE
|
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Supercoiled DNA
relaxation assay
|
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Alkaline elution assay |
|
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| Freeze at -80°C in working aliquots to
avoid repeated freeze-thawing. The enzyme is supplied in 20 mM Tris-HCl
(pH 8.0), 50 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1 mg/ml BSA, 50% glycerol. |
Products
|
4025-100-EB |
E. coli
Uracil-N-Glycosylase, 100 Units and 10X REC Buffer 6, 1 ml |
| Mouse Aag is a 36 kDa constitutively
expressed protein (1000-2000 copies/cell). It acts on 3-methyladenine,
3-methylguanine, 7-methylguanine, hypoxanthine, and a number of other
substrates. |
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| Purified from E. coli
containing a recombinant plasmid harboring the mouse Aag
protein. |
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| One unit cleaves 1 pmol of a
32P-labeled oligonucleotide probe containing hypoxanthine
within an oligonucleotide duplex in one hour at 37°C. |
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| 1X REC
Buffer 9 (10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 1 mM EDTA, 1 mM
EGTA, and 0.1 mM DTT), 4 pmoles 32P hypoxanthine
oligonucleotide, 4 pmoles complementary oligonucleotide, and
serial dilutions of enzyme in a reaction volume of 20 ΅l.
Incubate for 1 hour at 37°C. Reaction products are resolved by
20% denaturing PAGE. |
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| Store at -20°C.
For long-term storage freeze in working aliquots at -80°C to
avoid repeated freeze-thawing. The enzyme is stored in 10 mM
HEPES-KOH (pH 7.4); 100 mM KCl; 1 mM ETDA; 1 mM DTT; 0.2 mg/ml
BSA; 50% glycerol. |
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| Mouse Aag
catalyzes the excision of the following forms of DNA damage:
3-methyladenine, 3-methylguanine, 7-methylguanine, hypoxanthine,
1,N 6 ethanoadenine, and deoxyinosine. It may also function on
the following forms of DNA damage: 7- and 3-ethylpurines,
1-car-boxyethyladenine, 7-carboxyethylguanine, O 2
-methylpyrimidines, 7(2-ethoxyethyl)guanine,
7(2-hydroxyethyl)guanine, 7(2-chloroethyl)guanine,
1,2-bis(7-guanyl)ethane, 3-ethylth-ioethylpurines,
N2,3-ethenoguanine, N2,3-ethanoguanine, 5-hydroxymethyluracil,
5-formyl-uracil, 3,N 4 -ethenocytosine, 1,N 2 -ethenoguanine,
3,N 2 -ethenoguanine, chloroacetaldehyde cyclic adducts. |
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Products
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4090-100-EB |
Mouse
3-Methyladenine DNA Glycosylase, 100 Units and 10X REC Buffer 9, 1 ml |
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+32 (0) 16
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