Williams M V, Pollack J D
Department of Medical Microbiology and Immunology, Ohio State University, Columbus 43210.
J Bacteriol. 1990 Jun;172(6):2979-85. doi: 10.1128/jb.172.6.2979-2985.1990.
The DNA repair enzyme uracil-DNA glycosylase from Mycoplasma lactucae (831-C4) was purified 1,657-fold by using affinity chromatography and chromatofocusing techniques. The only substrate for the enzyme was DNA that contained uracil residues, and the Km of the enzyme was 1.05 +/- 0.12 microM for dUMP containing DNA. The product of the reaction was uracil, and it acted as a noncompetitive inhibitor of the uracil-DNA glycosylase with a Ki of 5.2 mM. The activity of the enzyme was insensitive to Mg2+, Mn2+, Zn2+, Ca2+, and Co2+ over the concentration range tested, and the activity was not inhibited by EDTA. The enzyme activity exhibited a biphasic response to monovalent cations and to polyamines. The enzyme had a pI of 6.4 and existed as a nonspherical monomeric protein with a molecular weight of 28,500 +/- 1,200. The uracil-DNA glycosylase from M. lactucae was inhibited by the uracil-DNA glycosylase inhibitor from bacteriophage PBS-2, but the amount of inhibitor required for 50% inhibition of the mycoplasmal enzyme was 2.2 and 8 times greater than that required to cause 50% inhibition of the uracil-DNA glycosylases from Escherichia coli and Bacillus subtilis, respectively. Previous studies have reported that some mollicutes lack uracil-DNA glycosylase activity, and the results of this study demonstrate that the uracil-DNA glycosylase from M. lactucae has a higher Km for uracil-containing DNA than those of the glycosylases of other procaryotic organisms. Thus, the low G + C content of the DNA from some mollicutes and the A.T-biased mutation pressure observed in these organisms may be related to their decreased capacity to remove uracil residues from DNA.
通过亲和色谱法和色谱聚焦技术，将来自莴苣支原体（831 - C4）的DNA修复酶尿嘧啶-DNA糖基化酶纯化了1657倍。该酶的唯一底物是含有尿嘧啶残基的DNA，对于含dUMP的DNA，该酶的Km为1.05±0.12微摩尔。反应产物是尿嘧啶，它作为尿嘧啶-DNA糖基化酶的非竞争性抑制剂，其Ki为5.2毫摩尔。在所测试的浓度范围内，该酶的活性对Mg2 +、Mn2 +、Zn2 +、Ca2 +和Co2 +不敏感，并且该活性不受EDTA抑制。该酶活性对单价阳离子和多胺表现出双相反应。该酶的pI为6.4，以非球形单体蛋白形式存在，分子量为28,500±1,200。来自莴苣支原体的尿嘧啶-DNA糖基化酶被噬菌体PBS - 2的尿嘧啶-DNA糖基化酶抑制剂抑制，但对该支原体酶产生50%抑制所需的抑制剂用量分别比引起大肠杆菌和枯草芽孢杆菌的尿嘧啶-DNA糖基化酶50%抑制所需的量高2.2倍和8倍。先前的研究报道一些柔膜菌缺乏尿嘧啶-DNA糖基化酶活性，本研究结果表明，来自莴苣支原体的尿嘧啶-DNA糖基化酶对含尿嘧啶DNA的Km高于其他原核生物的糖基化酶。因此，一些柔膜菌DNA的低G + C含量以及在这些生物体中观察到的A.T偏向的突变压力可能与其从DNA中去除尿嘧啶残基的能力降低有关。
