Highly Sensitive Radioactivity-Based DNA 3'-Phosphatase Activity Assay for Polynucleotide Kinase 3'-Phosphatase.
Highly Sensitive Radioactivity-Based DNA 3'-Phosphatase Activity Assay for Polynucleotide Kinase 3'-Phosphatase.
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基于放射性的高灵敏 DNA 3-磷酸酶活性测定，用于检测多核苷酸激酶 3-磷酸酶。
DOI：
10.1007/978-1-0716-3373-1_3
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Hazra,TapasK
中科院分区：
文献类型：
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作者：
Chakraborty,Anirban;Hazra,TapasK
内源性和外源性遗传毒性因子可在哺乳动物基因组中的链断裂位点处产生各种类型的不可连接的DNA末端。如果不修复，这种病变将阻碍转录和复制，并可能导致各种细胞病变。在各种“脏”DNA末端中，3′-磷酸是哺乳动物细胞中产生的最丰富的损伤之一。多核苷酸激酶3′-磷酸酶（PNKP）是哺乳动物细胞中分解3′-磷酸末端的主要DNA末端加工酶，参与DNA碱基切除修复（BER）、单链断裂修复和经典的非同源末端连接（C-NHEJ）介导的DNA双链断裂（DSB）修复。PNKP介导的3′-P加工后产生的3′-OH末端被DNA聚合酶用于填充差距，随后，缺口被DNA连接酶密封以完成修复过程。在这里，我们描述了两种新的检测系统，通过PNKP的3′-磷酸酶活性和PNKP介导的体外单链断裂修复与最小的修复成分（PNKP，DNA聚合酶和DNA连接酶）使用纯化的蛋白质或无细胞核提取物从哺乳动物细胞/组织中检测磷酸盐释放。这些检测方法具有高度的可重复性和灵敏度，研究人员将能够检测出患病哺乳动物细胞/组织中PNKP的3′-磷酸酶活性以及PNKP介导的单链断裂修复活性与正常健康对照的任何显著差异。
Endogenous and exogenous genotoxic agents can generate various types of non-ligatable DNA ends at the site of strand break in the mammalian genome. If not repaired, such lesions will impede transcription and replication and can lead to various cellular pathologies. Among various “dirty” DNA ends, 3′-phosphate is one of the most abundant lesions generated in the mammalian cells. Polynucleotide kinase 3′-phosphatase (PNKP) is the major DNA end-processing enzyme for resolving 3′-phosphate termini in the mammalian cells, and thus, it is involved in DNA base excision repair (BER), single-strand break repair, and classical nonhomologous end joining (C-NHEJ)-mediated DNA double-strand break (DSB) repair. The 3′-OH ends generated following PNKP-mediated processing of 3′-P are utilized by a DNA polymerase to fill in the gap, and subsequently, the nick is sealed by a DNA ligase to complete the repair process. Here we describe two novel assay systems to detect phosphate release by PNKP’s 3′-phosphatase activity and PNKP-mediated in vitro single-strand break repair with minimal repair components (PNKP, DNA polymerase, and DNA ligase) using either purified proteins or cell-free nuclear extracts from mammalian cells/tissues. These assays are highly reproducible and sensitive, and the researchers would be able to detect any significant difference in PNKP’s 3′-phosphatase activity as well as PNKP-mediated single-strand break repair activity in diseased mammalian cells/tissues vs normal healthy controls.