Starostenko Lidia V, Rechkunova Nadejda I, Lebedeva Natalia A, Lomzov Alexander A, Koval Vladimir V, Lavrik Olga I
Institute of Chemical Biology and Fundamental Medicine, Lavrentiev Av. 8, Novosibirsk, 630090, Russia; Department of Natural Sciences, Novosibirsk State University, 2 Pirogov Street, Novosibirsk, 630090, Russia.
Institute of Chemical Biology and Fundamental Medicine, Lavrentiev Av. 8, Novosibirsk, 630090, Russia.
DNA Repair (Amst). 2017 Feb;50:43-53. doi: 10.1016/j.dnarep.2016.12.007. Epub 2016 Dec 27.
The major enzyme in eukaryotic cells that catalyzes the cleavage of apurinic/apyrimidinic (AP or abasic) sites is AP endonuclease 1 (APE1) that cleaves the phosphodiester bond on the 5'-side of AP sites. We found that the efficiency of AP site cleavage by APE1 was affected by the benzo[a]pyrenyl-DNA adduct (BPDE-dG) in the opposite strand. AP sites directly opposite of the modified dG or shifted toward the 5' direction were hydrolyzed by APE1 with an efficiency moderately lower than the AP site in the control DNA duplex, whereas AP sites shifted toward the 3' direction were hydrolyzed significantly less efficiently. For all DNA structures except DNA with the AP site shifted by 3 nucleotides in the 3' direction (AP-BP-DNA), hydrolysis was more efficient in the case of (+)-trans-BPDE-dG. Using molecular dynamic simulation, we have shown that in the complex of APE1 with the AP-BP-DNA, the BP residue is located within the DNA bend induced by APE1 and contacts the amino acids in the enzyme catalytic center and the catalytic metal ion. The geometry of the APE1 active site is perturbed more significantly by the trans-isomer of BPDE-dG that intercalates into the APE1-DNA complex near the cleaved phosphodiester bond. The ability of DNA polymerases β (Polβ), λ and ι to catalyze gap-filling synthesis in cooperation with APE1 was also analyzed. Polβ was shown to inhibit the 3'→5' exonuclease activity of APE1 when both enzymes were added simultaneously and to insert the correct nucleotide into the gap arising after AP site hydrolysis. Therefore, further evidence for the functional cooperation of APE1 and Polβ in base excision repair was obtained.
真核细胞中催化脱嘌呤/脱嘧啶（AP或无碱基）位点切割的主要酶是AP核酸内切酶1（APE1），它能切割AP位点5'侧的磷酸二酯键。我们发现，APE1切割AP位点的效率受到互补链中苯并[a]芘-DNA加合物（BPDE-dG）的影响。与修饰的dG直接相对或向5'方向移动的AP位点被APE1水解，其效率略低于对照DNA双链体中的AP位点，而向3'方向移动的AP位点水解效率则显著降低。对于所有DNA结构，除了AP位点在3'方向移动3个核苷酸的DNA（AP-BP-DNA）外，（+）-反式-BPDE-dG情况下的水解效率更高。通过分子动力学模拟，我们表明在APE1与AP-BP-DNA的复合物中，BP残基位于由APE1诱导的DNA弯曲处，并与酶催化中心的氨基酸和催化金属离子接触。BPDE-dG的反式异构体插入到靠近切割的磷酸二酯键的APE1-DNA复合物中，对APE1活性位点的几何结构产生了更显著的扰动。还分析了DNA聚合酶β（Polβ）、λ和ι与APE1协同催化缺口填补合成的能力。结果表明，当同时添加这两种酶时，Polβ会抑制APE1的3'→5'核酸外切酶活性，并将正确的核苷酸插入AP位点水解后产生的缺口中。因此，获得了APE1和Polβ在碱基切除修复中功能协同的进一步证据。
