Colson P, Verly W G
Eur J Biochem. 1983 Aug 15;134(3):415-20. doi: 10.1111/j.1432-1033.1983.tb07583.x.
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.
大鼠肝细胞中的尿嘧啶-DNA糖基化酶大部分位于染色质中；然而，核液和细胞质中也有一些活性。染色质尿嘧啶-DNA糖基化酶已被纯化；该制剂不含内切核酸酶和外切核酸酶活性；该酶不需要二价阳离子，在pH约8时有较宽的最佳pH值，随着离子强度的增加和游离尿嘧啶的存在而受到强烈抑制。表观Km与含有尿嘧啶的DNA底物的链状无关，但单链底物的V略高。双链DNA中尿嘧啶取代的频率影响动力学参数：较高的频率会增加Km和V。当底物是双链而非单链时，NaCl和游离尿嘧啶的抑制作用更大。据推测，无论是作用于DNA还是酶，两者都阻碍了形成酶-底物复合物所需的双螺旋的打开。双链DNA中尿嘧啶残基频率较高时反应速率增加，这被解释为修复酶以连续方式工作的趋势。据推测，单链DNA也会发生连续性，而NaCl和游离尿嘧啶都会阻碍它，这解释了单链底物尿嘧啶含量较高时抑制作用更大的原因。
