Salas-Pacheco José M, Urtiz-Estrada Norma, Martínez-Cadena Guadalupe, Yasbin Ronald E, Pedraza-Reyes Mario
Institute of Investigation in Experimental Biology, Faculty of Chemistry, University of Guanajuato, Guanajuato 36060, Gto, Mexico.
J Bacteriol. 2003 Sep;185(18):5380-90. doi: 10.1128/JB.185.18.5380-5390.2003.
The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here. To this end, a recombinant YqfS protein containing an N-terminal His6 tag was synthesized in Escherichia coli and purified to homogeneity. An anti-His6-YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B. subtilis spores. The His6-YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3'-phosphatases. However, the purified protein lacked both 5'-phosphatase and exonuclease III activities. YqfS showed not only a high level of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules. Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3' blocking groups of DNA generated during both spore dormancy and germination.
本研究对枯草芽孢杆菌IV型脱嘌呤/脱嘧啶（AP）内切核酸酶同系物的酶学性质及其生理功能进行了研究，该同系物由yqfS编码，yqfS是一种在芽孢中特异性表达的基因。为此，在大肠杆菌中合成了含有N端His6标签的重组YqfS蛋白，并纯化至同质。抗His6 - YqfS多克隆抗体仅将YqfS定位在从枯草芽孢杆菌芽孢制备的细胞提取物中。His6 - YqfS蛋白表现出IV型DNA修复酶家族的酶学特性，如AP内切核酸酶和3'-磷酸酶。然而，纯化后的蛋白缺乏5'-磷酸酶和外切核酸酶III活性。YqfS不仅与大肠杆菌Nfo具有高度的氨基酸同一性，而且在含有AP位点的DNA（AP - DNA）存在的情况下，对EDTA失活具有高度抗性。这些结果表明，YqfS拥有一个三核锌中心，其中三个金属原子由九个保守的碱性残基和两个水分子紧密配位。电泳迁移率变动分析表明，YqfS具有使其能够结合和扫描未受损DNA以及与AP - DNA强烈相互作用的结构特性。yqfS能够在基因上互补缺乏主要AP内切核酸酶Nfo和外切核酸酶III的大肠杆菌突变体的DNA修复缺陷，这强烈表明其产物赋予细胞保护，使其免受氧化启动子和烷化剂的有害影响。因此，我们得出结论，枯草芽孢杆菌的YqfS是一种芽孢特异性蛋白，具有参与修复芽孢休眠和萌发过程中产生的DNA的AP位点和3'阻断基团所需的结构和酶学性质。
