Instituto de Biología Molecular Eladio Viñuela, CSIC, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Mol Microbiol. 2011 Jun;80(6):1657-66. doi: 10.1111/j.1365-2958.2011.07675.x. Epub 2011 May 12.
Uracil-DNA glycosylase (UDG) is a conserved DNA repair enzyme involved in uracil excision from DNA. Here, we report the biochemical characterization of UDG encoded by Bacillus subtilis, a model low G+C Gram-positive organism. The purified enzyme removes uracil preferentially from single-stranded DNA over double-stranded DNA, exhibiting higher preference for U:G than U:A mismatches. Furthermore, we have identified key amino acids necessary for B. subtilis UDG activity. Our results showed that Asp-65 and His-187 are catalytic residues involved in glycosidic bond cleavage, whereas Phe-78 would participate in DNA recognition. Recently, it has been reported that B. subtilis phage φ29 encodes an inhibitor of the UDG enzyme, named protein p56, whose role has been proposed to ensure an efficient viral DNA replication, preventing the deleterious effect caused by UDG when it eliminates uracils present in the φ29 genome. In this work, we also show that a φ29-related phage, GA-1, encodes a p56-like protein with UDG inhibition activity. In addition, mutagenesis analysis revealed that residue Phe-191 of B. subtilis UDG is critical for the interaction with φ29 and GA-1 p56 proteins, suggesting that both proteins have similar mechanism of inhibition.
尿嘧啶-DNA 糖基化酶（UDG）是一种保守的 DNA 修复酶，参与尿嘧啶从 DNA 中的切除。在这里，我们报告了枯草芽孢杆菌编码的 UDG 的生化特征，枯草芽孢杆菌是一种模型低 GC 革兰氏阳性生物体。纯化的酶优先从单链 DNA 中去除尿嘧啶，而不是双链 DNA，对 U:G 比对 U:A 错配的偏好更高。此外，我们已经确定了枯草芽孢杆菌 UDG 活性所必需的关键氨基酸。我们的结果表明，天冬氨酸 65 号和组氨酸 187 号是参与糖苷键断裂的催化残基，而苯丙氨酸 78 号则参与 DNA 识别。最近有报道称，枯草芽孢杆菌噬菌体 φ29 编码一种 UDG 酶抑制剂，称为蛋白 p56，其作用被认为是确保病毒 DNA 复制的效率，防止 UDG 消除 φ29 基因组中存在的尿嘧啶所造成的有害影响。在这项工作中，我们还表明，一种与 φ29 相关的噬菌体 GA-1 编码一种具有 UDG 抑制活性的 p56 样蛋白。此外，突变分析表明，枯草芽孢杆菌 UDG 的苯丙氨酸 191 残基对于与 φ29 和 GA-1 p56 蛋白的相互作用是关键的，这表明这两种蛋白具有相似的抑制机制。
