Intracellular localization of rat-liver uracil-DNA glycosylase. Purification and properties of the chromatin enzyme.
Most of the uracil-DNA glycosylase of the rat liver cell is located in chromatin; there is, however, some activity in the nuclear sap and in the cytoplasm. The chromatin uracil-DNA glycosylase has been purified; the preparation is devoid of endonuclease and exonuclease activities; the enzyme does not need divalent cations, has a broad optimum pH around 8, is strongly inhibited by increasing ionic strength and free uracil. The apparent Km is independent of the strandedness of the DNA substrate containing uracil, but V is slightly higher with the single-stranded substrate. The frequency of uracil substitution in the double-stranded DNA influences the kinetic parameters: a higher frequency increases both Km and V. The inhibitory effects of NaCl and free uracil are greater when the substrate is double-stranded rather than single-stranded. It is speculated that, acting either on the DNA or on the enzyme, both oppose the opening of the double helix necessary for the formation of the enzyme-substrate complex. The increased reaction rate with a higher frequency of uracil residues in double-stranded DNA is interpreted as a tendency for the repair enzyme to work in a processive way. It is supposed that processivity also occurs with single-stranded DNA and that it is opposed by both NaCl and free uracil, explaining a greater inhibition when the single-stranded substrate has a higher uracil content.
D000818:Animals; D001665:Binding Sites; D002467:Cell Nucleus / Q000201:enzymology; D055598:Chemical Phenomena; D002621:Chemistry; D002843:Chromatin / Q000201:enzymology*; D003600:Cytosol / Q000201:enzymology; D045647:DNA Glycosylases; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000201:enzymology*; D009699:N-Glycosyl Hydrolases / Q000302:isolation & purification*; D051381:Rats; D051981:Uracil-DNA Glycosidase
D002843:Chromatin; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase