Abstract
Abstract The incorporation of uracil into DNA in an in vitro repair reaction catalyzed by native Escherichia coli DNA polymerase I renders the newly synthesized uracil-containing portion of the resultant polymer susceptible to an endonucleolytic-like cleavage by the polymerase itself. This response is related to the presence of the 5' → 3' nuclease activity of this enzyme, since subtilisin-treated polymerase which lacks this nuclease activity also fails to attack uracil-containing DNA in this way.
Highlights
-+I11of the nondialyzable radioactive mntcrial in the thyminecontaining preparations was precipitable by this method. These findings suggested the presence of rat,her small oligonucleotidcs in the uracil-containing preparations
E. coli DK\‘h which had been 30y0 degraded by exonuclease and fully repaired rrith ITLlabeled nucleotides in reactions catalyzed by subtilisin-treated polymerase; this preparation contained
Based on tllc known +ecificitics of the nuclcascs associated with native DNA\ l~ol~mcrase I 3’ nuclease is inactive on small,singlc-atralltl fragments [19] and the 3’ + 5’ nuclease has a K, xvitll these substrates which is lo6 times greater than with native DNA [20]), the relative stability of the oligonucleotide peak and the apparent leveling off of acid solubilization in the uracil-containing reactions would be expected upon cow version of the substrate from long chain native polymers to small single-strand fragment.
Summary
DNA was repaired at 15” for 360 min, treated as described in the text to terminate the reaction and remove unincorporated nucleotides, and chromatographed on a column (1 X 58 cm) of Sephadex G-50 equilibrated with 0.5 3~ ammonium bicarbonate, pH 8.6-6 M urea. DNA polymerase in reactions containing dTTP (0) or dUTP (A).
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