多功能核酸酶APLF在DNA损伤修复中的作用与机制研究

DNA double-strand breaks(DSBs) are a major threat to genetic stability and hereditary defects in DSB repair result in a variety of disease pathologies.Loss of homologous recombination-mediated repair is associated with increased predisposition to cancer, including hereditary breast cancer,whereas loss of NHEJ is typically associated with immunodeficiency and neurological dysfunction.. Aprataxin-and-PNK-Like Factor (APLF; also denoted as PALF/C2orf13/XIP1)was identified as a novel structural component of the DSBs pathway, in mammalian cells. APLF is recruited to sites of DNA strand breakage via interaction of a C-terminal tandem zinc finger domain with poly (ADP-ribose), a nucleic acid like structure synthesized at sites of DNA breakage by poly(ADP-ribose)polymerases. Importantly, APLF appears to accelerate the rate of chromosomal NHEJ during the first few hours following γ-irradiation, suggesting that it is an accessory factor for NHEJ. The molecular mechanism by which APLF accelerates NHEJ is unclear. One possibility is that APLF is an end processing factor, because it has been reported to possess nuclease activity in vitro,and thus may process incompatible DNA termini. Another demonstrated that APLF depleted cells exhibit reduced levels of XRCC4 in chromatin, and that overexpression of XRCC4-Lig4 complex can circumvent the requirement for APLF for rapid rates of NHEJ, suggesting that APLF accelerates NHEJ primarily by promoting DNA ligase activity.It is possible that APLF promotes DNA ligation by modifying chromatin structure directly, since this protein interacts with histone H3/h4 and exhibits histone chaperone activity, in vitro. However, APLF also possesses an amino-terminal fork-head associated (FHA) domain that interacts directly with the XRCC4 and an MID domain that interacts directly with Ku heterodimer. But, the role and important of these interactions remains unknown. . This project aims to resolving the three dimensional structrue of APLF/XRCC1、APLF/XPCC4、APLF/H3/H4 and so on complexes by X-ray crystallographic method, combined with biochemistry, molecular biology research on function, to further explore the relationship between structure and function. Through these work, reveal the molecular architecture and action mechanism of APLF complexes, to further clarify the molecular mechanism of DNA recognize and cut, and lay the foundation for the APLF promotes chromatin disassembly and assembly in the naked DNA site.

多功能核酸酶APLF具有3’外切酶活性和单链DNA(ssDNA) 内切酶活性。其能与双链DNA损伤修复系统中的多种修复蛋白以及组蛋白相互作用，在DNA损伤修复、染色体拆分和重塑等过程中扮演极其重要的角色。因此，测定APLF及其复合物的结构和组装方式是研究其作用机理的根本。迄今为止，由于APLF及其复合物的蛋白样品制备和晶体生长非常困难，仅有APLF 锌指结构域的NMR结构被报道。本项目旨在通过X-ray 晶体学方法解析APLF/XRCC1、APLF/XPCC4、APLF/H3/H4等复合物的三维结构，并结合生物化学、分子生物学开展功能研究，进一步探讨结构和功能的关系。通过这些工作，揭示APLF复合物的分子组装与作用机制，阐明其识别和切割DNA损伤位点末端的分子机理，以及APLF在DNA损伤修复过程中调控组蛋白装配，促进染色体DNA的拆卸与重塑的作用机制。

生物体内的DNA结合蛋白在DNA损伤修复、染色体拆分和重塑等过程中扮演极其重要的角色。本课题展开对多功能核酸酶APLF，RadD以及SSBP等蛋白复合物三维结构和功能的关系的研究具有重要的生物学意义和创新性。.1．我们解析了多种DNA结合蛋白复合物的三维结构，并进行了功能实验和讨论，在高水平的国际期刊发表研究论文7篇。.2. 作为一种DNA损伤特异性组蛋白伴侣，APLF c -末端结构域能够与（H3-H4）2特异性作用，对APLF在体内DNA修复中的作用至关重要。然而，关于APLF与组蛋白或组蛋白之间相互作用的信息很少。因此，对APLF在DNA修复或其他生物过程中如何处理组蛋白的理解非常有限。这里，我们解析了人源APLF- ct和(H3-H4)2四聚体之间形成的复合物的晶体结构，生化分析表明，一个APLF分子与一个非对称(H3-H4)2异四聚体相互作用并使其稳定。值得注意的是，APLF与(H3-H4)2相互作用motif与一个重要的(H3-H4)2四聚体伴侣蛋白NAP1具有的组蛋白binding motif 高度同源，而且 APLF不同于其他已知的(H3-H4)伴侣蛋白，它们识别(H3-H4)2异源四聚体。尽管最近对(H3-H4)2四聚体及其组蛋白伴侣之间的相互作用进行了结构和生化分析，但(H3-H4)2四聚体是如何从四聚体中分离出来，并在邻近区域被保留，然后与DNA重新组装仍是一个谜。因此，这种新的伴侣蛋白APLF的histone-chaperone功能，以及与 (H3-H4)2四聚体在DNA修复过程中的结合方式揭示了一种新的组蛋白结构调控机制，并可能为其他生物学过程提供参考。工作已投稿。.3. DNA复制、转录和修复等多个生物学过程中都存在染色质结构的动态调控，并在表观遗传中发挥核心作用。在这些过程中，染色质组装配组蛋白沉积到DNA上或染色质卸载都通过组蛋白伴侣确保染色质结构得到最严格的修饰。 ALC1是具有依赖于pAR的染色质重构酶，其在染色质环境中促进DNA修复反应。APLF是ALC1-assciated复合物中一个主要的组织学结合成分，它与ALC1复合物中的H3-H4结合以协调染色质重构。目前，我们正在运用冷冻电镜技术，进一步解析APLF，ALC1等伴侣蛋白与核小体的复合物的原子分辨率三维结构。