Abstract Damaged DNA bases are removed by the base excision repair (BER) mechanism. This enzymatic process begins with the action of one of DNA glycosylases, which recognize damaged DNA bases and remove them by hydrolyzing N -glycosidic bonds with the formation of apurinic/apyrimidinic (AP) sites. Apurinic/apyrimidinic endonuclease 1 (APE1) hydrolyzes the phosphodiester bond on the 5′-side of the AP site with generation of the single-strand DNA break. A decrease in the functional activity of BER enzymes is associated with the increased risk of cardiovascular, neurodegenerative, and oncological diseases. In this work, we developed a fluorescence method for measuring the activity of key human DNA glycosylases and AP endonuclease in cell extracts. The efficacy of fluorescent DNA probes was tested using purified enzymes; the most efficient probes were tested in the enzymatic activity assays in the extracts of A549, MCF7, HeLa, WT-7, HEK293T, and HKC8 cells. The activity of enzymes responsible for the repair of AP sites and removal of uracil and 5,6-dihydrouracil residues was higher in cancer cell lines as compared to the normal HKC8 human kidney cell line.

中文翻译：

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使用荧光 DNA 探针测定细胞提取物中碱基切除修复酶的活性

摘要 碱基切除修复 (BER) 机制可去除受损的 DNA 碱基。该酶促过程始于其中一种 DNA 糖基化酶的作用，该酶识别受损的 DNA 碱基并通过水解 N-糖苷键并形成无嘌呤/无嘧啶 (AP) 位点来去除它们。无嘌呤/无嘧啶核酸内切酶 1 (APE1) 水解 AP 位点 5' 侧的磷酸二酯键，产生单链 DNA 断裂。BER 酶功能活性的降低与心血管、神经退行性和肿瘤疾病的风险增加有关。在这项工作中，我们开发了一种荧光方法，用于测量细胞提取物中关键的人类 DNA 糖基化酶和 AP 核酸内切酶的活性。使用纯化的酶测试荧光 DNA 探针的功效；在 A549、MCF7、HeLa、WT-7、HEK293T 和 HKC8 细胞提取物中的酶活性测定中测试了最有效的探针。与正常 HKC8 人肾细胞系相比，癌细胞系中负责修复 AP 位点和去除尿嘧啶和 5,6-二氢尿嘧啶残基的酶的活性更高。