Properties of purified uracil-DNA glycosylase from calf thymus. An in vitro study using synthetic DNA-like substrates.
The bovine uracil-DNA glycosylase previously isolated from thymocyte nuclei was further purified by 1 order of magnitude with the aid of affinity chromatography. The final preparation was totally devoid of DNase and apurinic or apyrimidinic (AP) endonuclease activities, and it corresponded to purifications of 457-fold over the nuclear extract and of about 2000-fold over the crude tissue homogenate. Most of the general enzyme properties already described were confirmed. Furthermore, this mammalian uracil-DNA glycosylase was shown to bind specifically with polymerized and not with monomeric nucleotide compounds, while having a preference for double-stranded forms. It cleaved N-glycosyl linkages only at the deoxyuridyl units located in internal positions of polynucleotide chains. The enzyme also used RNA-DNA hybrids as functional substrates and was practically ineffective on deoxyuridyl residues at the 3'-ends of nucleic acids. The activity of the glycosylase was greatly impaired in assays with DNA substrates that contained amounts of AP sites exceeding 5 microM. The inhibitory concentrations of AP residues were about 100 times lower than those found equally effective for the other reaction product, i.e. free uracil, and were almost comparable to the Km values for deoxyuridyl nucleotides in the DNA substrates. This all appears as a modulation of the glycosylase catalysis by the relative amounts of its substrate and product structures in DNA. The data lead us to surmise that the removal of uracil from cellular DNA is functionally coupled to the expected elimination of the formed AP sites by specific endonucleases. Base-exchange and base-insertion experiments with the purified enzyme yielded negative results under various conditions. The glycosylase behaved essentially as a hydrolase which has no associated base-insertase properties and irreversibly excises uracil from DNA by a mechanism for channeling the process to the next steps of the repair pathway.
D000818:Animals; D002250:Carbon Radioisotopes; D002417:Cattle; D045647:DNA Glycosylases; D004260:DNA Repair; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*; D014316:Tritium; D014498:Uracil / Q000494:pharmacology; D051981:Uracil-DNA Glycosidase
D002250:Carbon Radioisotopes; D014316:Tritium; D014498:Uracil; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase