Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, University of Vermont, Stafford Hall, 95 Carrigan Dr., Burlington, VT 05405-0068, United States.
DNA Repair (Amst). 2010 Feb 4;9(2):177-90. doi: 10.1016/j.dnarep.2009.11.008. Epub 2009 Dec 23.
The DNA glycosylases that remove oxidized DNA bases fall into two general families: the Fpg/Nei family and the Nth superfamily. Based on protein sequence alignments, we identified four putative Fpg/Nei family members, as well as a putative Nth protein in Mycobacterium tuberculosis H37Rv. All four Fpg/Nei proteins were successfully overexpressed using a bicistronic vector created in our laboratory. The MtuNth protein was also overexpressed in soluble form. The substrate specificities of the purified enzymes were characterized in vitro with oligodeoxynucleotide substrates containing single lesions. Some were further characterized by gas chromatography/mass spectrometry (GC/MS) analysis of products released from gamma-irradiated DNA. MtuFpg1 has substrate specificity similar to that of EcoFpg. Both EcoFpg and MtuFpg1 are more efficient at removing spiroiminodihydantoin (Sp) than 7,8-dihydro-8-oxoguanine (8-oxoG). However, MtuFpg1 shows a substantially increased opposite base discrimination compared to EcoFpg. MtuFpg2 contains only the C-terminal domain of an Fpg protein and has no detectable DNA binding activity or DNA glycosylase/lyase activity and thus appears to be a pseudogene. MtuNei1 recognizes oxidized pyrimidines on both double-stranded and single-stranded DNA and exhibits uracil DNA glycosylase activity. MtuNth recognizes a variety of oxidized bases, including urea, 5,6-dihydrouracil (DHU), 5-hydroxyuracil (5-OHU), 5-hydroxycytosine (5-OHC) and methylhydantoin (MeHyd). Both MtuNei1 and MtuNth excise thymine glycol (Tg); however, MtuNei1 strongly prefers the (5R) isomers, whereas MtuNth recognizes only the (5S) isomers. MtuNei2 did not demonstrate activity in vitro as a recombinant protein, but like MtuNei1 when expressed in Escherichia coli, it decreased the spontaneous mutation frequency of both the fpg mutY nei triple and nei nth double mutants, suggesting that MtuNei2 is functionally active in vivo recognizing both guanine and cytosine oxidation products. The kinetic parameters of the MtuFpg1, MtuNei1 and MtuNth proteins on selected substrates were also determined and compared to those of their E. coli homologs.
能够切除氧化 DNA 碱基的 DNA 糖苷酶可分为两大类：Fpg/Nei 家族和 Nth 超家族。根据蛋白质序列比对，我们在结核分枝杆菌 H37Rv 中鉴定出了 4 种推定的 Fpg/Nei 家族成员和 1 种推定的 Nth 蛋白。我们使用实验室创建的双顺反子载体成功地表达了所有 4 种 Fpg/Nei 蛋白。MtuNth 蛋白也以可溶性形式过表达。使用含有单损伤的寡脱氧核苷酸底物在体外对纯化酶的底物特异性进行了表征。一些酶通过对 γ 射线照射 DNA 释放的产物进行气相色谱/质谱（GC/MS）分析进一步进行了表征。MtuFpg1 的底物特异性与 EcoFpg 相似。EcoFpg 和 MtuFpg1 均比 7,8-二氢-8-氧鸟嘌呤（8-oxoG）更有效地去除螺环亚氨基二氢嘧啶（Sp）。然而，MtuFpg1 与 EcoFpg 相比，表现出明显增强的相反碱基鉴别能力。MtuFpg2 仅包含 Fpg 蛋白的 C 末端结构域，没有检测到 DNA 结合活性或 DNA 糖苷酶/裂解酶活性，因此似乎是一个假基因。MtuNei1 识别双链和单链 DNA 上的氧化嘧啶，并表现出尿嘧啶 DNA 糖苷酶活性。MtuNth 识别多种氧化碱基，包括尿素、5,6-二氢尿嘧啶（DHU）、5-羟基尿嘧啶（5-OHU）、5-羟胞嘧啶（5-OHC）和甲基乙内酰脲（MeHyd）。MtuNei1 和 MtuNth 均切除胸腺嘧啶二醇（Tg）；然而，MtuNei1 强烈偏爱（5R）异构体，而 MtuNth 仅识别（5S）异构体。MtuNei2 作为重组蛋白在体外没有表现出活性，但与在大肠杆菌中表达的 MtuNei1 一样，它降低了 fpg mutY nei 三突变体和 nei nth 双突变体的自发突变频率，表明 MtuNei2 在体内识别鸟嘌呤和胞嘧啶氧化产物时具有功能活性。还测定了 MtuFpg1、MtuNei1 和 MtuNth 蛋白在选定底物上的动力学参数，并与它们的大肠杆菌同源物进行了比较。
