Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Lavrentyev Ave, Novosibirsk 630090, Russian Federation.
Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences, 8 Lavrentyev Ave, Novosibirsk 630090, Russian Federation; Department of Natural Sciences, Novosibirsk State University, 2 Pirogova St, Novosibirsk 630090, Russian Federation.
Biochimie. 2018 Sep;152:53-62. doi: 10.1016/j.biochi.2018.06.012. Epub 2018 Jun 26.
Apurinic/apyrimidinic endonuclease Apn1 of Saccharomyces cerevisiae is known as a key player of the base excision DNA repair (BER) pathway in yeast. BER is initiated by DNA glycosylases, whereas Apn1 can start DNA repair individually in the nucleotide incision repair (NIR) pathway. The aim of this research was to elucidate kinetic and structural dynamic aspects of Apn1 involvement in the NIR process. One of the key characteristics of AP endonuclease's interactions is known to be divalent metal ions playing a part of a cofactor. Well-studied human APE1 employs Mg ions, with metal ion concentration's affecting enzymatic activity exerted by APE1. In our study, we aimed to test the effect of the Mg ion on Apn1's NIR catalysis by examining structural dynamics of DNA during the interaction in real time using the stopped-flow technique. To test NIR activity of Apn1, deoxyribooligonucleotide duplexes containing a 5,6-dihydro-2'-deoxyuridine (DHU) residue were employed as substrates. A 2-aminopurine (2-aPu) residue was a reporter group fluorescence intensity of which was detected during Apn1-DNA interactions. NIR activity of both WT and H83A Apn1 was found to be arrested during the interaction with a DNA duplex containing the 2-aPu residue upstream of DHU. We conducted molecular dynamics simulations to elucidate the structural features of complexes of the enzyme with DHU-containing DNAs. The NIR recruiting S. cerevisiae Apn1 proceeds via multistep rearrangements of the complex of Apn1 with a DHU-containing DNA substrate and results in the incised product of the reaction. For wild-type Apn1, the catalytic rate constants do not depend on the Mg concentration, i.e., they are equal in NIR and BER buffers, with equilibrium association constant K being 10-fold higher in NIR buffer. Our data reveal more delicate regulation of Apn1's NIR activity due to the more complicated kinetic mechanism, as compared to BER.
酵母的脱嘌呤/脱嘧啶内切核酸酶 Apn1 是碱基切除 DNA 修复 (BER) 途径中的关键因子。BER 由 DNA 糖苷酶起始，而 Apn1 可以在核苷酸切除修复 (NIR) 途径中单独启动 DNA 修复。本研究旨在阐明 Apn1 参与 NIR 过程的动力学和结构动态特征。AP 内切核酸酶相互作用的一个关键特征是二价金属离子作为辅助因子的一部分。研究较多的人类 APE1 利用 Mg 离子，金属离子浓度影响 APE1 的酶活性。在我们的研究中，我们旨在通过使用停流技术实时检查 DNA 相互作用过程中的结构动态，测试 Mg 离子对 Apn1 的 NIR 催化作用。为了测试 Apn1 的 NIR 活性，使用含有 5,6-二氢-2'-脱氧尿嘧啶 (DHU) 残基的脱氧核糖寡核苷酸双链作为底物。2-氨基嘌呤 (2-aPu) 残基是报告基团，在 Apn1-DNA 相互作用过程中检测其荧光强度。发现 WT 和 H83A Apn1 的 NIR 活性在与含有 DHU 的 DNA 双链相互作用时被阻止，该 DNA 双链中的 2-aPu 残基位于 DHU 的上游。我们进行了分子动力学模拟，以阐明酶与含 DHU 的 DNA 复合物的结构特征。酵母 Apn1 的 NIR 招募过程通过 Apn1 与含 DHU 的 DNA 底物复合物的多步重排进行，并导致反应的切口产物。对于野生型 Apn1，催化速率常数不依赖于 Mg 浓度，即在 NIR 和 BER 缓冲液中相等，在 NIR 缓冲液中的平衡结合常数 K 高 10 倍。与 BER 相比，我们的数据揭示了 Apn1 的 NIR 活性受到更复杂的动力学机制的更精细调控。
