|
| |
UV Damage Enzymes
Ultraviolet DNA
Endonuclease (UVDE)
E. coli Photolyase
T4 Endonuclease V (T4-PDG)
Chlorella Virus
Pyrimidine Dimer Glycosylase
Products
|
4100-100-EB |
UVDE
Enzyme, 100 µl and 10X REC Buffer 5, 1 ml |
| E. coli
photolyase repairs cyclobutane pyrimidine dimers (CPD) in UV irradiated
DNA by a reaction in which light energy drives electron transport from a
catalytic chromophore, reduced FADH, to the pyrimidine dimer, leading to
its photolysis. The enzyme is a monomeric protein of 471 amino acids (MW
53,994 daltons). It includes two noncovalently attached cofactors, the
blue light harvest cofactor MTHF and the catalytic cofactor FADH-
. Photolyase selectively binds to the CPD in a light-independent step.
The MTHF cofactor absorbs a blue light photon and then excites FADH-
by energy transfer. The excited FADH-, *FADH-,
transfers an electron to split the CPD to regenerate the pyrimidines.
The electron is transferred back to the photolyase, and the intact DNA
dissociates. |
| |
| Recombinant E. coli photolyase is
purified from an E. coli strain harboring the photolyase phr
gene. |
| |
| One unit is the amount
of enzyme required to repair 100 ng of a supercoiled plasmid irradiated
with 50 mJ of 254 nm UV light in 1 hour at 37°C during photoreactivation
by 365 nm blue light at the rate of 2 mW/cm. The repair is measured as a
loss in conversion of the supercoiled plasmid to relaxed, open circle
form following treatment with the CPD-specific endonuclease T4-PDG. |
| |
| Reactions of 20 µl
contain 250 ng of supercoiled plasmid irradiated with 50 mJ of 254 nm UV
light, 1X REC™ Buffer 14 (20 mM
Tris-Cl (pH 7.8), 1 mM EDTA, 1 mM DTT, 50 mM NaCl), and photolyase.
Reactions are set up in yellow light to minimize activation of the
photolyase. The enzyme is photoactivated by irradiation with 365 nm
light at a fluence of 2 mW/cm and the reaction is allowed to proceed for
1 hour at 37°C. 5 units of T4-PDG are added and the tubes are incubated
for 1 hour at 37°C. Supercoiled and relaxed forms of the plasmid are
resolved by 1% agarose gel electrophoresis and detected by ethidium
bromide staining. |
| |
| Studies involving UV-induced DNA damage |
| |
| Freeze in working
aliquots at -80°C to avoid repeated freeze-thawing. The enzyme is
supplied in a buffer containing 20 mM Tris-Cl (pH 7.8), 1 mM EDTA, 50%
glycerol, 50 mM NaCl, and 1 mM DTT. |
Products
|
4145-100-EB |
E. coli
Photolyase, 100 Units and 10X REC™ Buffer 14, 1ml |
|
4145-500-EB |
E. coli Photolyase,500 Unit and 10X REC™ Buffer 14, 1ml |
| T4 Endonuclease V is a DNA glycosylase of
MW 16 kDa with an associated AP lyase activity that is specific for the
cis-syn isomer of cyclobutane pyrimidine dimers induced by UV
irradiation. |
| |
| Purified from E. coli containing a
recombinant plasmid harboring the T4 phage DenV gene. |
| |
| One unit is the amount of enzyme required
to completely relax 250 ng of a UV-irradiated supercoiled plasmid in 30
minutes at 37°C. |
| |
| 1X REC™
Buffer 11 (25 mM NaPO4 (pH 6.8), 1 mM EDTA, 100 mM NaCl, 1 mM
DTT, 0.1 mg/ml BSA) supercoiled plasmid (250 ng) irradiated with 100 J/m2
UV light, and serial dilutions of enzyme in a 20 µl reaction volume are
incubated for 30 minutes at 37°C. |
| |
| • |
FLARE™
 |
| • |
Supercoiled DNA
relaxation assay |
|
| |
| Store at 4°C. The enzyme is supplied in 25
mM NaPO4 (pH 6.8), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1
mg/ml BSA. |
Products
|
4055-100-EB |
T4
Endonuclease V (T4-Pyrimidine Dimer Glycosylase/T4-PDG), 100,000 Units
and 10X REC™ Buffer 11, 1 ml |
| cv-PDG is a DNA
glycosylase of MW 16 kDa with an associated AP lyase activity that is
equally specific for the cis-syn and trans-syn isomers of cyclobutane
pyrimidine dimers induced by UV irradiation. |
| |
| Purified from E. coli containing a
recombinant plasmid harboring the Paramecium bursaria Chlorella virus
pdg gene. |
| |
| One unit is the amount of enzyme required
to completely relax 250 ng of a UV-irradiated supercoiled plasmid in 30
minutes at 37°C. |
| |
| 1X REC™
Buffer 11 (25 mM NaPO4 (pH 6.8), 1 mM EDTA, 1 mM EDTA, 100 mM
NaCl, 1 mM DTT, 0.1 mg/ml BSA), supercoiled plasmid (250 ng) irradiated
with 100 J/m2 UV light, and serial dilutions of enzyme in a
20 µl reaction volume are incubated for 30 minutes at 37°C. |
| |
| • |
FLARE™
|
| • |
Supercoiled DNA
relaxation assay |
|
| |
| Store at 4°C. The enzyme is supplied in 25
mM NaPO4 (pH 6.8), 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1
mg/ml BSA. |
Products
|
4065-100-EB |
Chorella
Virus Pyrimidine Dimer (cv-PDG), 1000 Units and 10X REC™ Buffer 11, 1 ml |
|
+32 (0) 16
58 90 45 
+32 (0) 16
50 90 45