A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase.
A mollicute (mycoplasma) DNA repair enzyme: purification and characterization of uracil-DNA glycosylase.
复制标题
软体（支原体）DNA 修复酶：尿嘧啶-DNA 糖基化酶的纯化和表征。
DOI：
10.1128/jb.172.6.2979-2985.1990
复制
发表时间：
1990
影响因子：
3.2
通讯作者：
Pollack,JD
中科院分区：
文献类型：
--
作者：
Williams,MV;Pollack,JD
利用亲和层析和层析聚焦技术，将来自乳糖支原体（831-C4）的DNA修复酶尿嘧啶-DNA糖基化酶纯化了1，657倍。该酶的唯一底物是含有尿嘧啶残基的DNA，并且对于含有dUMP的DNA，该酶的Km为1.05 +/-0.12 microM。反应产物为尿嘧啶，对尿嘧啶-DNA糖基化酶具有非竞争性抑制作用，Ki值为5.2 mM. The酶活性对Mg ~（2+）、Mn ~（2+）、Zn ~（2+）、Ca ~（2+）和Co ~（2+）不敏感，EDTA对酶活性无抑制作用。酶的活性表现出双相响应一价阳离子和多胺。该酶的pI为6.4，以非球形单体蛋白存在，分子量为28，500 +/-1，200。尿嘧啶-DNA糖基化酶。乳酸菌被来自噬菌体PBS-2的尿嘧啶-DNA糖基化酶抑制剂抑制，但是使支原体酶抑制50%所需的抑制剂的量分别是使来自大肠杆菌和枯草芽孢杆菌的尿嘧啶-DNA糖基化酶抑制50%所需的抑制剂的量的2.2倍和8倍。以往的研究报道，一些柔膜菌缺乏尿嘧啶-DNA糖基化酶活性，本研究结果表明，柔膜菌尿嘧啶-DNA糖基化酶活性较低。与其他原核生物的糖基化酶相比，莴苣对含尿嘧啶的DNA具有更高的Km。因此，一些柔膜菌DNA的低G + C含量和在这些生物体中观察到的A. T偏向突变压力可能与它们从DNA中去除尿嘧啶残基的能力降低有关。
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.