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#4100-100-EB S. pombe UVDE Enzyme & Buffer

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  Price : 479   EUR
544   USD
371   GBP
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542   CHF

Product name : S. pombe UVDE Enzyme & Buffer

Catalog number : 4100-100-EB

Quantity: 100 ul

Availability: Yes

Supplier name : Trevigen

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Description: Exposure to UV irradiation can damage DNA through the formation of bipyrimidine DNA photoproducts that are potentially cytotoxic and mutagenic. The two major classes of dimeric photoproducts are cyclobutane pyrimidine dimers and (6-4)-photoproducts (see figure 1).
Ultraviolet Damage Endonuclease (UVDE) is a nuclease involved in the repair of bipyrimidine DNA photoproducts and acts as an alternative to base or nucleotide excision repair pathways.
Repair of pyrimidine dimers by UVDE-mediated excision occurs by cleavage immediately 5’ to the photoproduct site on the DNA strand in an ATPindependent manner, and is more efficient in vitro than
nucleotide excision repair.
Source: Trevigen’s UVDE is a fusion protein consisting of glutathione-S-transferase fused to a truncated form of the UVDE protein from Schizosaccharomyces pombe (GST-Δ228-UVDE). Unlike full length UVDE, which has proved to be unstable and difficult to solubilize, GST-Δ228-UVDE lacks 228 amino acids from the amino terminus (of UVDE) and is soluble and stable when stored at -80°C. The presence of the N-terminal deletion does not affect the native properties of the protein, and optimal activity occurs at 30°C.

Substrate Specificity: Enzymatic studies revealed that UVDE from Schizosaccharomyces pombe recognizes pyrimidine dimers, photoproducts, apurinic/apyrimidinic sites, uracil, dihydrouracil, and other non-UV-induced DNA adducts.1,2 Biochemical and genetic analysis also suggest that UVDE may be involved in orchestrating mismatch repair in vivo, and this enzyme is also active on insertion-deletion loops.
Storage Buffer: 50 mM Tris-Cl (pH 6.0), 10 mM glutathione, and 10% glycerol.
Storage Conditions: Store at -80 oC. For long term storage, freeze in working aliquots at -80 oC to avoid repeated freeze-thawing.
Assay Conditions & Analysis: UVDE treatment of supercoiled DNA, containing adducts such as cyclobutane pyrimidine dimers, leads to relaxation of the supercoiling resulting in a relative shift in electrophoretic mobility. This may be visualized in agarose gels by staining with an intercalating dye such as ethidium bromide. Qualitative evaluation is performed by comparing the relative amounts of DNA contained within bands corresponding to supercoiled DNA (ccc) and to nicked (relaxed) forms. There are other more sensitive dyes available (e.g. SYBR® Gold, (Molecular Probes)), which reduce the amount of DNA required per lane. The actual concentrations of DNA and enzyme, and incubation times may have to be adjusted for optimal results. For increased sensitivity, or if a specific gene or DNA strand is under investigation, the DNA may be transferred by Southern blot to a membrane and probed with
radiolabeled DNA fragments from the DNA strand or gene of interest.


Materials and Reagents:
30oC Incubator Test and Control DNA
Agarose Gel Electrophoresis Equipment and Pipettors and tips
Reagents Ice Bath
UV transilluminator 10 mg/ml EtBr in water


Method:
1. Prepare the test DNA at approximately 250 ng/μl in water. In assays utilizing other forms of DNA, e.g. mitochondrial DNA, the concentration of DNA may need to be increased. Avoid exposure of the DNA to UV and solar light during preparation and storage. Always run controls to check for damage that may be introduced during DNA preparation and handling.
2. For a 20 μl reaction mix combine the following on ice in microcentrifuge tubes:

3. Incubate all tubes at 30°C for 15 to 60 minutes.
4. Add 2 μl of 10X EDTA Loading Buffer (see appendix) to each tube.
5. Resolve DNA by agarose gel electrophoresis (e.g. use 1.0% Trevigel 5000 (cat#9806-050-P)).
6. Stain gel for 15 minutes in 0.5 μg/ml ethidium bromide in water.
7. Visualize by viewing under ultraviolet light.
Interpretation of results: By agarose gel electrophoresis, supercoiled plasmid DNA migrates faster than either nicked or linear DNA. The different migration patterns of the DNA forms and relative DNA content in each band allows for interpretation of conversion from supercoiled to nicked and/or linear DNA. Figure 2 illustrates relaxation of supercoiling by GST-Δ228-UVDE on plasmid DNA exposed to UV.

A 3.2 kb purified plasmid was treated with or without 1000 J/m2 UV at 254 nm. Approximately 1 μg of DNA was treated with or without 250 ng of GST-Δ228-UVDE for 45 minutes at 30°C and analyzed by electrophoresis on a 1 % Trevigel 5000 gel in 1X TAE buffer. The gel was stained with 0.5 μg/ml ethidium bromide for 15 minutes then photographed under ultraviolet light. GST-Δ228-UVDE does not exhibit activity on unmodified DNA (lane 2) and UV treatment alone does not alter the ratio of the DNA forms of the plasmid (lane 3). Treatment of UV irradiated DNA with GST-Δ228-UVDE converted the covalently closed circular plasmid to nicked and linear forms.

Note: When smaller quantities of DNA are treated with UVDE, some nicking activity may occur in control DNA reflecting UV damage in plasmid preparation.

Buffer Composition:
1. 10X RECTM Buffer 5: 200 mM HEPES pH 6.5; 100 mM MgCl2; 10 mM MnCl2; 1M NaCl
2. 10X EDTA Loading Buffer: 25% Ficoll; 2% SDS; 10 mM EDTA pH 8.0
3. 0.5 μg/ml ethidium bromide: Prepare a 10 mg/ml stock solution of ethidium bromide in water. Store in a dark or foil covered bottle at room temperature.
Dilute 10 mg/ml stock solution 1 in 2000 in water, sufficient for gel immersion. The diluted stock may be reused several times if stored in the dark. Dispose of according to state and federal regulations.

 

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Pathways :
WP142: mRNA processing
WP1493: Carbon assimilation C4 pathway
WP2340: Thiamine (vitamin B1) biosynthesis and salvage
WP2341: vitamin B1 (thiamin) biosynthesis and salvage pathway
WP2344: vitamin B6 (pyridoxine, pyridoxal, pyridoxamine) biosynthesis and salvage pathway
WP2349: vitamin B3 (niacin), NAD and NADP biosynthesis pathway
WP626: Abscisic Acid Biosynthesis

Related Genes :
[uve1 uvde SPBC19C7.09c] UV-damage endonuclease (UVDE) (EC 3.-.-.-)
[pch1 SPBC32F12.06] Cyclin pch1 (Pombe cyclin C homolog 1)
[rhp6 ubc2 SPAC18B11.07c] Ubiquitin-conjugating enzyme E2 2 (EC 2.3.2.23) (E2 ubiquitin-conjugating enzyme 2) (RAD6 homolog) (Ubiquitin carrier protein 2) (Ubiquitin-protein ligase 2)
[prr1 SPAC8C9.14] Transcription factor prr1 (Pombe response regulator 1)
[pku80 ku80 SPBC543.03c] ATP-dependent DNA helicase II subunit 2 (EC 3.6.4.12) (ATP-dependent DNA helicase II subunit Ku80) (Pombe Ku80)
[ubc4 SPBC119.02] Ubiquitin-conjugating enzyme E2 4 (EC 2.3.2.23) (E2 ubiquitin-conjugating enzyme 4) (Ubiquitin carrier protein 4) (Ubiquitin-protein ligase 4)
[ptr3 SPBC1604.21c SPBC211.09] Ubiquitin-activating enzyme E1 1 (EC 6.2.1.45) (Poly(A)+ RNA transport protein 3)
[atg7 SPBC6B1.05c] Ubiquitin-like modifier-activating enzyme atg7 (ATG12-activating enzyme E1 atg7) (Autophagy-related protein 7)
[fas1 SPAC926.09c] Fatty acid synthase subunit beta (EC 2.3.1.86) [Includes: 3-hydroxyacyl-[acyl-carrier-protein] dehydratase (EC 4.2.1.59); Enoyl-[acyl-carrier-protein] reductase [NADH] (EC 1.3.1.9); [Acyl-carrier-protein] acetyltransferase (EC 2.3.1.38); [Acyl-carrier-protein] malonyltransferase (EC 2.3.1.39); S-acyl fatty acid synthase thioesterase (EC 3.1.2.14)]
[plh1 SPBC776.14] Phospholipid:diacylglycerol acyltransferase (PDAT) (EC 2.3.1.158) (Pombe LRO1 homolog 1)
[ubp9 SPBC1703.12] Probable ubiquitin carboxyl-terminal hydrolase 9 (EC 3.4.19.12) (Deubiquitinating enzyme 9) (Ubiquitin thioesterase 9) (Ubiquitin-specific-processing protease 9)
[ubp21 ubp15 ubpd SPBC713.02c] Ubiquitin carboxyl-terminal hydrolase 21 (EC 3.4.19.12) (Deubiquitinating enzyme 21) (Ubiquitin thioesterase 21) (Ubiquitin-specific-processing protease 21)
[ubc7 ubcp3 SPBP16F5.04] Ubiquitin-conjugating enzyme E2-18 kDa (EC 2.3.2.23) (E2 ubiquitin-conjugating enzyme 7) (Ubiquitin carrier protein) (Ubiquitin-protein ligase)
[ubc13 spu13 SPAC11E3.04c] Ubiquitin-conjugating enzyme E2 13 (EC 2.3.2.23) (E2 ubiquitin-conjugating enzyme 13) (Ubiquitin carrier protein 13) (Ubiquitin-protein ligase 13)
[egt1 mug158 SPBC1604.01 SPBC1677.01c] Ergothioneine biosynthesis protein 1 (Meiotically up-regulated gene 158 protein) [Includes: L-histidine N(alpha)-methyltransferase (EC 2.1.1.44); Hercynylcysteine S-oxide synthase (EC 1.14.99.51)]
[abc2 SPAC3F10.11c] ATP-binding cassette transporter abc2 (ABC transporter abc2) (EC 3.6.3.-) (ATP-energized glutathione S-conjugate pump abc2) (Glutathione S-conjugate-transporting ATPase abc2)
[psk1 SPCC4G3.08] Serine/threonine-protein kinase psk1 (EC 2.7.11.1) (Ribosomal S6 kinase homolog psk1) (S6K homolog psk1)
[ubp4 SPBC18H10.08c] Probable ubiquitin carboxyl-terminal hydrolase 4 (EC 3.4.19.12) (Deubiquitinating enzyme 4) (Ubiquitin thioesterase 4) (Ubiquitin-specific-processing protease 4)
[gsa1 gsh2 SPAC3F10.04] Glutathione synthetase large chain (GSH synthetase large chain) (GSH-S) (EC 6.3.2.3) (Glutathione synthase large chain) (Phytochelatin synthetase)
[hus5 ubc9 SPAC30D11.13] SUMO-conjugating enzyme ubc9 (EC 2.3.2.-) (Ubiquitin carrier protein 9) (Ubiquitin carrier protein hus5) (Ubiquitin-conjugating enzyme E2-18 kDa)
[rpn1 mts4 SPBP19A11.03c] 26S proteasome regulatory subunit rpn1 (19S regulatory cap region of 26S protease subunit 2) (Proteasome non-ATPase subunit mts4)
[fas2 lsd1 SPAC4A8.11c] Fatty acid synthase subunit alpha (EC 2.3.1.86) (p190/210) [Includes: Acyl carrier; 3-oxoacyl-[acyl-carrier-protein] reductase (EC 1.1.1.100) (Beta-ketoacyl reductase); 3-oxoacyl-[acyl-carrier-protein] synthase (EC 2.3.1.41) (Beta-ketoacyl synthase)]
[glo1 SPBC12C2.12c SPBC21D10.03c] Lactoylglutathione lyase (EC 4.4.1.5) (Aldoketomutase) (Glyoxalase I) (Glx I) (Ketone-aldehyde mutase) (Methylglyoxalase) (S-D-lactoylglutathione methylglyoxal lyase)
[sty1 hog1 phh1 spc1 SPAC24B11.06c] Mitogen-activated protein kinase sty1 (MAP kinase sty1) (EC 2.7.11.24) (MAP kinase spc1)
[nth1 SPAC30D11.07] Endonuclease III homolog (EC 3.2.2.-) (EC 4.2.99.18) (Bifunctional DNA N-glycosylase/DNA-(apurinic or apyrimidinic site) lyase) (DNA glycosylase/AP lyase)
[dna2 SPBC16D10.04c] DNA replication ATP-dependent helicase/nuclease dna2 [Includes: DNA replication nuclease dna2 (EC 3.1.-.-); DNA replication ATP-dependent helicase dna2 (EC 3.6.4.12)]
[rad4 cut5 SPAC23C4.18c] S-M checkpoint control protein rad4 (P74) (Protein cut5)
[atg5 mug77 SPBC4B4.10c] Autophagy protein 5 (Meiotically up-regulated gene 77 protein)
[ubc11 ubcp4 SPCC1259.15c] Ubiquitin-conjugating enzyme E2-20 kDa (EC 2.3.2.23) (E2 ubiquitin-conjugating enzyme 11) (Ubiquitin carrier protein) (Ubiquitin-protein ligase)
[hnt3 SPCC18.09c] Aprataxin-like protein (EC 3.1.11.7) (EC 3.1.12.2) (Hit family protein 3)

Bibliography :
[29335541] Modulation of UVB-induced Carcinogenesis by Activation of Alternative DNA Repair Pathways.
[26547444] Immuno-capture of UVDE generated 3'-OH ends at UV photoproducts.
[24039606] The Uve1 endonuclease is regulated by the white collar complex to protect cryptococcus neoformans from UV damage.
[21493688] Cdt1 proteolysis is promoted by dual PIP degrons and is modulated by PCNA ubiquitylation.
[19152795] Damage recognition by UV damage endonuclease from Schizosaccharomyces pombe.
[12531030] Two budding yeast RAD4 homologs in fission yeast play different roles in the repair of UV-induced DNA damage.
[11202359] Bound transcription factor suppresses photoproduct formation in the NF-kappa B promoter.
[10924509] Cellular responses and repair of single-strand breaks introduced by UV damage endonuclease in mammalian cells.
[10801329] Ultraviolet damage endonuclease (Uve1p): a structure and strand-specific DNA endonuclease.
[10766807] Alternative excision repair pathway of UV-damaged DNA in Schizosaccharomyces pombe operates both in nucleus and in mitochondria.