Krokan H, Wittwer C U
Nucleic Acids Res. 1981 Jun 11;9(11):2599-613. doi: 10.1093/nar/9.11.2599.
Uracil-DNA glycosylase was partially purified from HeLa cells. Various substrates containing [3H]dUMP residues were prepared by nick-translation of calf thymus DNA. The standard substrate was double-stranded DNA with [3H]dUMP located internally in the chain. Compared to the release of uracil from this substrate, a 3-fold increase in the rate was seen with single-stranded DNA, and a 20-fold reduction in the rate was observed when the [3H]dUMP-residue was located at the 3'end. The rate of [3H]uracil release decreased progressively when one, two or three of the dNMP residues were replaced by the corresponding rNMP; in the extreme case when the substrate contained [3H]dUMP in addition to rCMP, rGMP, and rAMP, the rate of [3H]uracil release was less than 3% of that of the control. The enzyme was inhibited to the same extent by uracil and the uracil analogs 6-aminouracil and 5-azauracil, but very weakly, or not at all, by 5 other analogs. Our results suggest strongly that uracil-DNA glycosylase has a high degree of selectivity for uracil in dUMP residues located internally in DNA chains and that the recognition of the correct substrate also depends on the residues flanking dUMP being deoxyribonucleotides.
尿嘧啶-DNA糖基化酶从人宫颈癌（HeLa）细胞中部分纯化得到。通过小牛胸腺DNA的切口平移制备了各种含有[³H]dUMP残基的底物。标准底物是双链DNA，其中[³H]dUMP位于链内部。与从该底物释放尿嘧啶相比，单链DNA的释放速率提高了3倍，而当[³H]dUMP残基位于3'端时，释放速率降低了20倍。当一个、两个或三个dNMP残基被相应的rNMP取代时，[³H]尿嘧啶的释放速率逐渐降低；在极端情况下，当底物除了含有rCMP、rGMP和rAMP外还含有[³H]dUMP时，[³H]尿嘧啶的释放速率不到对照的3%。该酶受到尿嘧啶以及尿嘧啶类似物6-氨基尿嘧啶和5-氮杂尿嘧啶的同等程度抑制，但受到其他5种类似物的抑制作用非常微弱或根本没有抑制作用。我们的结果强烈表明，尿嘧啶-DNA糖基化酶对位于DNA链内部的dUMP残基中的尿嘧啶具有高度选择性，并且对正确底物的识别还取决于dUMP侧翼的残基为脱氧核糖核苷酸。
