| Source: |
Purified from E. coli containing a recombinant plasmid harboring the S. solfataricus Dpo4 gene.
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| Unit Definition: |
One unit of enzyme incorporates 1 nmole of dTMP onto the 3' end of d(T)24 using poly(dA)300 as a template in 30 minutes at 60°C.
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| Assay Conditions & Analysis: |
1X REC Buffer 15 [10 mM HEPES-NaOH (pH 7.8), 1 mM DTT, 50 mM NaCl, 100 µg/ml BSA, 0.1% Triton X-100], 10 mM MgCl2, 1 pmole of 32P-labeled d(T)24, 2 pmole of poly(dA)300, and serial dilutions of enzyme in a 20 µl reaction volume are incubated for 30 minutes at 60°C. Reaction products are resolved by 20% denaturing polyacrylamide gel electrophoresis. Bands are cut out and radioactivity counted to quantify newly synthesized products.
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| Specificity: |
Dpo4 catalyzes translesion synthesis across the following forms of DNA damage: AP sites, cis-syn thymine-thymine dimer, 6-4 photoproducts, cisplatinum- and acetyl aminofluorene guanine adducts.
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| Components: |
Part # |
Component |
Size |
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3900-500-15 |
10 x REC Buffer 15 |
1 ml |
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4150-010-02 |
100 mM MgCl2 |
1 ml |
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4150-010-03 |
50 mM MnCl2 |
1 ml |
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| Applications: |
Amplification of damaged DNA. |
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| Storage: |
Store at –20°C in a manual defrost freezer. For long-term storage, freeze at –80°C in working aliquots. Avoid repeated freeze-thawing. Enzyme may be diluted in 1X REC Buffer 15 for immediate use. |
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| References: |
1. Boudsocq, F, Iwai, S, Hanaoka, F, and Woodgate, R. 2001. Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4): an archaeal DinB-like DNA polymerase with lesion-bypass properties akin to eukaryotic poln. Nucleic Acids Res. 29, 4607–4616.
2. McDonald JP, Hall A, Gasparutto D, Cadet J, Ballantyne J, Woodgate R. Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs. Nucleic Acids Res. 2006 34:1102-11.
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