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Mismatch Repair Enzymes
E. coli Endonuclease V
E. coli MutY Enzyme
E. coli
Mismatch Uracil DNA Glycosylase (Mug Protein)
Thermostable TDG Protein
| Ionizing radiation, UV
light, or nitrous acid can generate A/T to C/G transitions in DNA via
the premutagenic base, hypoxanthine, or through the deamination of
adenine. Hypoxanthine is removed in E. coli by 3-methyladenine
DNA glycosylase, but not Endonuclease V. It is believed that
Endonuclease V requires accessory proteins to remove the damaged base.
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| Purified from E. coli containing a
recombinant plasmid harboring the E. coli Endonuclease V gene. |
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| One unit is the amount
of enzyme required to cleave 1 pmoles of a 32P-labeled
oligonucleotide probe in one hour at 37°C at a C/C mismatch when
hybridized to a complementary oligonucleotide. |
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| 1X REC™
Buffer 2 (20 mM HEPES-NaOH (pH 7.4), 100 mM KCl, 2 mM MnCl2
and 0.1 mg/ml BSA), 4 pmoles of 32P-oligonucleotide probe, 4
pmoles of complementary oligonucleotide (which generate a C/C mismatch
when hybridized together) and serial dilutions of enzyme in a 20 µl
reaction volume are incubated for 1 hour at 37°C. Cleavage products are
resolved by 20% denaturing PAGE. |
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| Endonuclease V
recognizes mismatches in duplex DNA and cleaves the second and third
phosphodiester bonds 3’ to the mismatch at 95% and 5% frequency,
respectively, in the strand with the mismatch closest to the 5’ end. The
opposite strand containing the mismatch closest to its 3’ end is also
cleaved, but the extent of cleavage varies depending on the mismatch.
Both strands are cleaved if the mismatch is located in the middle of the
oligonucleotide duplex and cleavage occurs about equally at the second
and third phosphodiester bonds 3’ from the mismatch. Endonuclease V also
cleaves DNA duplexes containing inosine, deoxyuridine, AP sites, urea
residues, hairpin or unpaired loops, flap, and pseudo-Y structures. |
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| E. coli Endonuclease V Kit |
| Cat# |
Component |
Size |
4035-500-01
|
Endonuclease V
|
500 Units |
| 3900-500-02
|
10X REC™
Buffer 2 |
1 ml |
| 4019-1 |
REC™
Water
|
1 ml |
| 4017-500 |
3X REC™
Alkali Loading Buffer |
500 µl |
| 3800-100-01
|
MUA Oligonucleotide
|
100 pmol |
| 3800-100-05
|
MLA Oligonucleotide
|
100 pmol |
| 3800-100-06
|
MLT Oligonucleotide
|
100 pmol |
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| MIDAScan™AT
Kit
|
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| Freeze at -80°C in
working aliquots to avoid repeated freeze-thaws. The enzyme is supplied
in 20 mM Trisc-Cl (pH 7.8), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 50% (v/v)
glycerol. |
Products
|
4035-500-EB |
E. coli
Endonuclease V, 500 Units and 10X REC™ Buffer 2, 1 ml |
|
4035-500-K |
E. coli
Endonuclease V Kit, 500 Units |
| E. coli Mut Y acts together
with Fpg to prevent the potentially mutagenic consequences of 8-oxo-dG
lesions. The 8-oxo-dG lesions that escape repair by Fpg frequently pair
with A during DNA replication, producing an 8-oxo-dG:A mispair. Mut Y
removes the A from this base pair to initiate base excision repair. In
the absence of Mut Y, DNA replication past the 8-oxo-dG:A mismatch
results in thymine incorporation opposite the adenine in one of the
daughter strands, creating a fixed mutation. Mut Y has an associated AP
lyase activity. |
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|
|
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| Purified from E. coli
containing a recombinant plasmid harboring the E. coli
MutY gene. |
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| One unit is the amount of enzyme
required to cleave 1 pmole of an oligonucleotide duplex
containing an A/G mismatch in 1 hour at 37°C. Only the strand
with the A is cleaved. |
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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 µl.
Incubate for 1 hour at 37°C. Reaction products are resolved by
20% denaturing PAGE. |
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| • |
MIDAS™
 |
| • |
MIDAScan™
|
| • |
Mismatch
cleavage assays |
|
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| Cat# |
Componenet |
Size |
4000-500-01
|
Mut Y
|
500 Units
|
| 3900-500-04
|
10X REC™
Buffer 4 |
1 ml
|
| 4019-1
|
REC™
Water |
1 ml
|
| 4017-500
|
3X Alkaline
Loading Buffer |
500 µl
|
| 3800-100-01
|
MUA
Oligonucleotide |
100 pmol
|
| 3800-100-05
|
MLA
Oligonucleotide |
100 pmol
|
| 3800-100-06
|
MLT
Oligonucleotide |
100 pmol
|
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| Freeze in working aliquots at -20°C
in a manual defrost freezer to avoid repeated freeze-thawing.
Enzyme is supplied in 10 mM HEPES-KOH (pH 7.4), 100 mM KCl, 1 mM
EDTA, 0.1 mg/ml BSA, and 50%(v/v) glycerol. |
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| MutY DNA glycosylase recognizes A/G
and A/8-oxo-dG mismatches in duplex DNA and cleaves the strand
containing the A. The opposite strand is not cleaved. |
|
Products
|
4000-500-EB |
E. coli
MutY Enzyme, 500 Units and 10X REC Buffer 4, 1 ml |
|
4000-500-K |
E. coli MutY Kit, 500 Units |
| E. coli Mug is
an 18 kDa constitutively expressed protein. It has been proposed that
Mug is responsible for the removal of 3,N4-ethenocytosine
from DNA, although the biological role remains unclear. The Mug protein
excises 3,N4-ethenocytosine and removes the uracil base from
mismatches in the order of U:G>U:A. |
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| Purified from E.
coli containing a recombinant plasmid encoding the E. coli
Mug protein. |
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| One unit cleaves 1 pmole
of a 32P-labeled oligonucleotide probe containing 3,N4-ethenocytosine
within an oligonucleotide duplex in one hour at 37°C. |
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| 1X REC Buffer 6 (20 mM
Tris-Cl (pH 8.0), 0.1 mg/ml BSA, 1 mM EDTA, and 1 mM DTT), 1 pmole 3,N4-ethenocytosine
oligonucleotide (Cat# 3864-100-01) labeled with 32P, 1 pmole
complement D (Cat# 3849-100-04), and serial dilutions of enzyme in a
reaction volume of 20 µl, incubated for 1 hour at 37°C. |
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| E. coli Mug
catalyzes the excision of 3,N4-ethenocytosine, a form of DNA
damage, in double or single stranded DNA. It also acts to excise uracil
in uracil-guanine mismatches. |
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| Freeze in working
aliquots at -20°C in a manual defrost freezer to avoid repeated
freeze-thawings. The enzyme is supplied in 25 mM HEPES (pH 7.6), 0.5 mM
EDTA, 1 mM DTT, 1 mM PMSF, and 10% (v/v) glycerol. |
Products
|
4125-100-EB |
E. coli
Mismatch Uracil DNA Glycosylase (Mug protein), 100 Units and 10X REC™
Buffer 6, 1 ml |
| TDG is a thermostable
thymine DNA glycosylase from Methanobacterium thermoau-totrophicum. The
optimal temperature for the enzyme is 65°C. The enzyme lacks significant
AP lyase or endonuclease activity. TDG works effectively in heteroduplex
analysis to detect T/G and G/G mismatches. |
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| Thermostable TDG is
purified from E. coli containing a recombinant plasmid
harboring the thermostable TDG gene. |
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| One unit is the amount
of enzyme required to cleave 1 pmole of an oligonucleotide duplex
containing a T/G mismatch in 1 hour at 65°C. Only the strand containing
the T is cleaved. |
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| 1X REC Buffer 4 (10 mM
HEPES-KOH (pH 7.4), 100 mM KCl, and 10 mM EDTA), 4 pmoles of each oligo
in the T/A Control Thermophilic Oligonucleotide Set (Cat# 3810-100-TA:
TUT=TDG probe, TLA=complementary strand, and TLG=negative control), and
serial dilutions of enzyme in a 20 µl reaction volume are incubated for
1 hour at 65°C. |
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| TDG enzyme recognizes
T/G, mismatches in duplex DNA and cleaves the strand with the T. The
opposite strand is not cleaved. The enzyme also recognizes G/G
mismatches and cleaves one strand or the other. TDG enzyme exhibits poor
AP endonuclease and AP lyase activities. |
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| • |
MIDAS™
|
| • |
MIDAScan™
|
| • |
Mismatch detection |
|
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| Freeze in working
aliquots at -20°C to avoid repeated freeze-thawing. The enzyme is
supplied in 10 mM HEPES KOH, pH 7.4, 100 mM KCl, 1 mM EDTA, 0.1 mg/ml
BSA, and 50% glycerol. |
Products
|
4070-500-EB |
Thermostable TDG Protein, 500 Units and 10X REC Buffer 4, 1 ml |
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