Samples have been stored at ?80 C till required AGT concentratio

Samples had been stored at ?80 C right up until required. AGT concentrations had been measured spectrophotometrically working with ?280 three 104 M?1cm?one . DNA samples Oligonucleotides of sixteen, 24 and 26 residues have been purchased from Invitrogen. The had been purified from the supplier utilizing reverse phase HPLC, and immediately after receipt, by phenol extraction followed by ether extraction and intensive dialysis against 10mM Tris buffer. Concentrations have been measured spectrophotometrically implementing extinction coefficients provided by the makers. The 24 mer oligonucleotide E was labeled at its five hydroxyl with 32P as described . Unincorporated ATP was removed by buffer exchange employing Sephadex G 10 mini spin columns equilibrated with 10 mM Tris , 1 mM EDTA. DNA duplexes have been prepared by mixing purified five labeled oligonucleotide with 1.
05 fold molar purchase AMG-517 excesses of complementary unlabeled strands , heating to 90 C for one min, then slowly cooling to area temperature. Just after annealing, the purities of duplex DNAs have been tested by native polyacrylamide gel electrophoresis . Mobility shift assays Binding reactions have been carried out at 20 C in ten mM Tris , 50 mM KCl and 5 mM 2 mercaptoethanol. Mixtures were equilibrated for 30 min. Duplicate samples incubated for longer intervals gave identical results, indicating that equilibrium had been attained . Electrophoresis was carried out in 20 polyacrylamide gels, as described . Autoradiographic images had been captured on storage phosphor screens detected which has a Typhoon phosphorimager and quantitated with Picture Quant computer software . DNA alkyltransferase assays The NarI endonuclease is inactive when substrate DNA includes an O6 methylguanine at position 2 of its cognate sequence .
Cleavage is restored by DNA alkyltransferase action offered by AGT. Oligonucleotides C and D containing the NarI sequence had been annealed separately with complementary oligo E, as described over. Alkyltransferase reactions Polydatin have been carried out for ten or 15 min with 1.one M AGT and 0.25 M duplex DNA dissolved in twenty mM Tris acetate , 50 mM potassium acetate, 10 mM magnesium acetate, 1 mM dithiothreitol. Reactions have been stopped by addition of SDS followed by two extractions with water saturated phenol and 3 extractions with water saturated diethylether. Samples were incubated at area temperature under vacuum to evaporate ether in advance of digestion with NarI. Samples had been resolved by native gel electrophoresis ; electrophoretic distributions have been recorded and quantified using a phosphorimager.
In reactions carried out in AGT excess, 98 of the DNA in our preparations was a substrate for the two alkyltransferase and NarI activity. When this DNA was titrated with AGT, maximal repair was obtained when one.04, corresponding to an alkyltransferase activity of 96 .