人类Pif1解旋酶解旋G4 DNA分子机制的研究

G-quadruplexes(G4 DNA) are four-stranded non-canonical nucleic acids structures formed in specific tandem guanine rich sequence. Accumulating evidences now point towards the existence of these structures in biologically functional regions of many genomes and telomeres. They provides a variety of regulatory functions in processes such as DNA replication, cellular senescence and tumorigenesis. Therefore, organisms have evolved highly specific helicases to resolve G4s structures. Human Pif1 helicase (hPif1) catalyzed-G4 DNA unwinding will have important impact on G4s biology functions, but it remains largely unknown about how hPif1 helicase resolves G4 DNA at molecular level. Based on the previous study, we intend to explore the enzymatic properties of hPif1 helicase underlying the recognition and specific binding of G4 DNA, the binding and unwinding kinetics between hPif1 and G4s, and the movement of hPif1 and conformational change of G4 at single molecule level for the first time. By integrating many new multidisciplinary methods, such as the molecular modeling of homologous protein and site-directed mutagenesis, dynamic light scattering, fluorescence polarization, the stopped-flow spectrophotometry and the single-molecule FRET assays, we will depthy study the detailed G4s’ unfolding mechanism mentioned above. Achieving the proposal goals, will be important for us to deeply understand the molecular mechanism of hPif1-catalyzed G4 DNA unwinding, and also coud provide new perspectives for the early molecular events and molecular targets of cancers in the futher research.

G-四链体DNA (G4 DNA) 是由富含重复鸟嘌呤序列折叠形成的四链形态DNA高级结构；其广泛存在于基因组和端粒中，对DNA复制、细胞衰老、肿瘤发生等生命过程都起重要的调节作用。生物体进化出许多解旋酶都可以特异解旋G4 DNA，其中人类Pif1解旋酶（hPif1）介导G4 DNA解旋效应对其生物学功能将会产生重要影响。但是hPif1解旋G4 DNA的分子机制仍尚未明确。本项目在前期研究基础上，综合运用同源蛋白分子模拟和定点突变、动态激光散射、荧光偏振分析、快速停流荧光检测、单分子荧光能量共振转移等技术，首次系统研究hPif1识别与特异结合G4 DNA的酶学特性、与G4 DNA相互反应的结合动力学和解旋动力学过程、解链G4过程中G4 DNA的分子行为与hPif1的分子运动轨迹，以期阐明hPif1解旋G4 DNA的分子机制，为后期进一步研究G4 DNA相关的肿瘤疾病的早期分子事件提供新的视角。

G4 DNA是广泛存在于基因组和端粒中的、由重复鸟嘌呤序列折叠形成的四链形态DNA高级结构，而人类Pif1解旋酶（hPif1）可特异性催化G4 DNA解旋，对其生物学功能产生重要影响；但是hPif1解旋G4 DNA的分子机制仍尚未明确。本课题综合运用快速停流荧光检测、单分子荧光监测平台、荧光偏振分析、动态激光散射等技术，首次较为系统研究hPif1解旋G4 DNA的结合反应酶学特性、hPif1催化G4 DNA解旋动力学特征参数以及解链G4 DNA的单分子运动过程。研究结果表明，只有昆虫-杆状病毒胞内表达与多层次纯化才能获得纯度高、活性好的hPif1蛋白；其结合反应最佳底物是5’-尾链的wt Tel26 G4（Kd≈8.5）；hPif1解旋含G4 DNA底物的最佳条件是30℃,Tris-HCl pH7.5, 20mM NaCl/KCl, 2 mM MgCl2，2 mM DTT，且hPif1核心结构域蛋白解旋活性达到全长蛋白的85%，N-端截短蛋白无明显解旋活性却存在较强的退火活性；hPif1不具有解旋DNA-RNA杂交链的倾向性；本研究首次提出端粒G4 DNA应是hPif1最佳G4 DNA底物（后续单分子实验得以验证）；首次发现G4 DNA激活hPif1解旋dsDNA活性，且并通过解旋特征参数、结合反应动力学、DLS等揭示其原因是hPif1结合ss-G4 DNA底物为二聚状态；单分子FERT技术首次发现hPif1催化G4 DNA解旋与再折叠是往复过程，且存在解旋中间体；hPif1解旋G4 DNA过程是在结合位点与解旋位点间反复跳跃的单分子运动行为——由此基本阐明hPif1解旋G DNA底物的动力学特征与单分子作用机制。此外，本项目还资助hPif1其他同源蛋白解旋反应分子特征的研究以拓展了本项目的横向对比；其他分子靶标检测新方法研究则为后续诊疗肿瘤相关G4 DNA奠定基础。