Abstract
A DNA glycosylase activity that excises oxidized, fragmented thymine residues from a polydeoxyribonucleotide has been purified 9,500-fold to apparent homogeneity from Escherichia coli. The purified enzyme also excises thymine glycol and cleaves DNA at apurinic sites, and appears to be identical with E. coli DNA endonuclease III. The enzyme catalyzes the release of several different forms of oxidized thymine, including urea, methyltartronylurea and 5-hydroxy-5-methylhydantoin. The molecular weight of the native protein is 25,000, and the same value is obtained for the denatured homogeneous protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Highlights
LKB Products, heparin-Sepharose CL-GB from Pharmacia, Affi-Gel blue (100-200 mesh) from Bio-Rad, and radioactive compounds from Amersham
Prior to use in enzyme assays, all oxidized polymers crucial reversion site, detects oxidative mutagens much more were mixedwith an equimolar amount of poly(dT) to generate doubleefficiently than previously used tester strains with critically placed G .C base pairs(9).As part of a search forDNA repair enzymes that might act to counteract oxidation damage of DNA, we have previously described a DNA glycosylase activity which catalyzes the release of urea
Size and Subunit Structure-The urea-DNA glycosylase activity waspurified 9500-fold from E. coli extracts.The preparation (Fraction VI) appeared tobe homogeneous, since only a singleprotein band was visualized by silver staining of SDS-polyacrylamide gels (Fig. 2)
Summary
Way, except thatthe polymer substrate contained scattered, "Clabeled thymine glycol residues or methyltartronylurea residues in Materials and Reference Coumpounds-Single-stranded DNA cel- the poly(dA) strand instead of urea residues In all these polymers, lulose was prepared according to Alberts and Herrick (12).Phospho- about 50% of the radioactive material could be released under conditions of enzyme excess. The urea-DNA glycosylase activity was found exclusively in the 0.6 M NaCl fraction, while several other DNAglycosylases (uracil-, 3methyladenine I-, and 3-methyladenine 11-)eluted a t lower salt concentrations and thuswere largely removedT. Gel Filtration-Fraction I11 was applied to a column of Ultrogel AcA-54 (1.6 X 150 cm) equilibrated with buffer B (15 mM potassium phosphate, pH 7.4, 7 mM 2-mercaptoethanol, 1 mM EDTA, 5% glycerol) containing 0.5 M NaCl. The enzymatically active fractions eluted more slowly than the majority of the applied protein (Fig. 1). Fox Chase Cancer Center, Philadelphia, PA) were prepared as above
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
