Molecular Biology Program, Sloan-Kettering Institute, New York, New York, USA
J Bacteriol. 2018 Aug 10;200(17). doi: 10.1128/JB.00151-18. Print 2018 Sep 1.
5'- and 3'-end healing are key steps in nucleic acid break repair in which 5'-OH and 3'-PO or 2',3'-cyclic-PO ends are converted to 5'-PO and 3'-OH termini suitable for sealing by polynucleotide ligases. Here, we characterize HD-Pnk as a bifunctional end-healing enzyme composed of N-terminal HD (histidine-aspartate) phosphoesterase and C-terminal P-loop polynucleotide kinase (Pnk) domains. HD-Pnk phosphorylates 5'-OH DNA in the presence of ATP and magnesium. HD-Pnk has 3'-phosphatase and 2',3'-cyclic-phosphodiesterase activity in the presence of transition metals, optimally cobalt or copper, and catalyzes copper-dependent hydrolysis of -nitrophenylphosphate. HD-Pnk is encoded by the LIG-PARG-HD-Pnk three-gene operon, which includes polynucleotide ligase and poly(ADP-ribose) glycohydrolase genes. We show that whereas HD-Pnk is inessential for growth, its absence sensitizes by 80-fold bacteria to killing by 9 kGy of ionizing radiation (IR). HD-Pnk protein is depleted during early stages of post-IR recovery and then replenished at 15 h, after reassembly of the genome from shattered fragments. ΔHD-Pnk mutant cells are competent for genome reassembly, as gauged by pulsed-field gel electrophoresis. Our findings suggest a role for HD-Pnk in repairing residual single-strand gaps or nicks in the reassembled genome. HD-Pnk-Ala mutations that ablate kinase or phosphoesterase activity sensitize to killing by mitomycin C. End healing is a process whereby nucleic acid breaks with "dirty" 3'-PO or 2',3'-cyclic-PO and 5'-OH ends are converted to 3'-OH and 5'-PO termini that are amenable to downstream repair reactions. is resistant to massive doses of ionizing radiation (IR) that generate hundreds of dirty DNA double-strand breaks and thousands of single-strand breaks. This study highlights HD-Pnk as a bifunctional 3'- and 5'-end-healing enzyme that helps protect against killing by IR. HD-Pnk appears to act late in the process of post-IR recovery, subsequent to genome reassembly from shattered fragments. HD-Pnk also contributes to resistance to killing by mitomycin C. These findings are significant in that they establish a role for end-healing enzymes in bacterial DNA damage repair.
5'端和 3'端修复是核酸断裂修复的关键步骤，其中 5'端 -OH 和 3'端 -PO 或 2'，3'环-PO 末端被转化为适合多核苷酸连接酶封闭的 5'端-PO 和 3'端-OH 末端。在这里，我们将 HD-Pnk 鉴定为一种具有双功能末端修复酶，由 N 端 HD（组氨酸-天冬氨酸）磷酸酯酶和 C 端 P 环多核苷酸激酶（Pnk）结构域组成。在 ATP 和镁存在下，HD-Pnk 磷酸化 5'端-OH DNA。在过渡金属存在下，HD-Pnk 具有 3'磷酸酶和 2'，3'环磷酸二酯酶活性，最适为钴或铜，并催化 -硝基苯膦酸的铜依赖性水解。HD-Pnk 由多核苷酸连接酶和聚（ADP-核糖）糖水解酶基因组成的 LIG-PARG-HD-Pnk 三基因操纵子编码。我们表明，虽然 HD-Pnk 对 生长不是必不可少的，但它的缺失使细菌对 9kGy 电离辐射（IR）的杀伤敏感 80 倍。在 IR 后早期恢复过程中，HD-Pnk 蛋白耗尽，然后在基因组从破碎片段重新组装后 15 小时补充。ΔHD-Pnk 突变体细胞能够进行基因组重组，如脉冲场凝胶电泳所示。我们的研究结果表明，HD-Pnk 在修复重组基因组中的残留单链缺口或缺口方面发挥作用。使激酶或磷酸酯酶活性失活的 HD-Pnk-Ala 突变使 对丝裂霉素 C 的杀伤敏感。末端修复是一种过程，其中具有“脏”3'端 -PO 或 2'，3'环-PO 和 5'端 -OH 末端的核酸断裂被转化为适合下游修复反应的 3'端-OH 和 5'端-PO 末端。对产生数百个脏 DNA 双链断裂和数千个单链断裂的大量电离辐射（IR）具有抗性。本研究强调了 HD-Pnk 作为一种具有双功能 3'端和 5'端修复酶的作用，有助于防止 IR 杀伤。HD-Pnk 似乎在 IR 后恢复过程的后期起作用，在基因组从破碎片段重新组装之后。HD-Pnk 还有助于抵抗丝裂霉素 C 的杀伤。这些发现意义重大，因为它们确立了末端修复酶在细菌 DNA 损伤修复中的作用。
