Institute of Chemical Biology and Fundamental Medicine (ICBFM), Siberian Branch of Russian Academy of Sciences, Novosibirsk 630090, Russia.
Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St., Novosibirsk 630090, Russia.
Molecules. 2018 Aug 21;23(9):2101. doi: 10.3390/molecules23092101.
Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5' to the damage. In addition to the AP-endonuclease activity, APE1 possesses 3'-5' exonuclease activity, which presumably is responsible for cleaning up nonconventional 3' ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3'-end nucleotide removal in the 3'-5' exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5' dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3' end of the short oligonucleotide. The impact of the 3'-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3'-5' exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3' nucleotide removal by APE1 in the 3'-5' exonuclease process is the release of the detached nucleotide from the enzyme's active site.
人类嘌呤糖苷酶/嘧啶糖苷酶（AP）内切酶 1（APE1）是参与 DNA 损伤修复的关键酶之一。APE1 的主要作用是通过催化磷酸二酯键在损伤部位 5'端的水解切割，启动 AP 位点的修复。除了 AP 内切酶活性外，APE1 还具有 3'-5'外切酶活性，该活性可能负责清除由于 DNA 损伤或 DNA 修复途径中的转换中间体而产生的非传统 3'末端。在本研究中，在预稳态条件下研究了 APE1 催化的 3'-5'外切酶过程中 3'末端核苷酸去除的动力学机制。DNA 底物为具有一个 5' 悬垂末端的脱氧核苷酸双链体，其在短寡核苷酸的 3' 末端的第 1、2、4 或 6 位处含有荧光 2-氨基嘌呤残基。研究了 3' 末端核苷酸（包含错配、未受损碱基或修饰碱基以及无碱基位点或磷酸基团）对 3'-5' 外切酶活性效率的影响。动力学数据表明，APE1 在 3'-5' 外切酶过程中从酶的活性位点释放脱落核苷酸是 3' 核苷酸去除的限速步骤。
