Abstract
Escherichia coli photolyase is a DNA repair enzyme which monomerizes pyrimidine dimers, the major UV photoproducts in DNA, to pyrimidines in a light-dependent reaction. We recently described the construction of a tac-phr plasmid that greatly overproduces the enzyme (Sancar, G. B., Smith, F. W., and Sancar, A. (1983) Nucleic Acids Res. 11, 6667-6678). Using a strain carrying the overproducing plasmid as the starting material, we have developed a purification procedure that yields several milligrams of apparently homogeneous enzyme. The purified protein is a single polypeptide that has an apparent Mr of 49,000 under both denaturing and nondenaturing conditions. The enzyme has no requirement for divalent cations and it restores the biological activity of irradiated DNA only in the presence of photoreactivating light. The purified photolyase has a turnover number of 2.4 dimers/molecule/min; this value agrees well with the in vivo rate of photoreactivation in E. coli.
Highlights
Buffers-The cell lysis buffer contained 50 mM Tris-HCI, pH 8.0, dure that yields several milligrams of apparently ho- 100 mM NaCI, 1 mM EDTA, 10 m M P-mercaptoethanol
Fiber Optic Scanner Model 800 (Kontes Scientific Instruments) and Cyclobutadipyrimidines or pyrimidine dimers are tmheajor DNA photoproducts produced by ultraviolet (200-300 nm) radiation
If left unrepaired pyrimidine dimerscan cause the peaks were integrated with a Model 3390A Integrator from Hewlett Packard
Summary
Bacterial Strainsand Plasmids-E. coli CSR603 (recA1 uurA6 p h r l ) was obtained from the E. coli GeneticStock Center, Yale. After 2 h of outgrowth for polyacrylamide gels appears as the most prominent cellular protein This enabledus to follow the purification of the enzyme by analyzing the variouspurificationfractionson SDS-polyacrylamide gels. From 5 liters of culture, 17 g of cells were obtained by centrifugation; the cells expression of antibiotic resistance, triplicate samples were plated on were resuspended in 100 ml of lysis buffer and frozen in a dry. From the change in transformation efficiency as a result of photoreactivation, the amount the cells the suspension was thawed at 0 "Covernight and the of repaired pyrimidine dimers was calculated. By centrifugation at 32,000 x g for 20 min and a1t 20,000 x g for 1 h to obtain a clear cell-free extract (Fraction 1, 100 ml)
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