The Role of the Human Holliday Junction Resolvase Component SLX4 in DNA Recombina

描述（由申请人提供）：该培训项目将在哈佛医学院Wade Harper博士的实验室进行博士后水平的培训。这项研究估计需要3年才能完成，将重点放在最近发现的Holliday结分解元件SLX4上。这是研究DNA重组修复的一个很好的系统。哈珀博士的实验室和哈佛医学院都有许多技术，这些技术将极大地促进拟议的实验研究，如蛋白质组学和显微镜成像设备。本研究的第一个目的是研究SLX4定位和激活的调控机制，以及SLX4在端粒维持中的作用。由于翻译后修饰是参与损伤反应的蛋白质定位的重要因素，SLX4及其相关因子将在这些过程的背景下进行研究。质谱方法将用于鉴定SLX4上在没有和存在DNA损伤的情况下磷酸化的残基。这些残基将发生突变，以测试去除特定残基上的SLX4磷酸化是否会影响其对损伤位点的招募，或影响其HJ分解活性。SLX4包含两个串联泛素相互作用基序，因此将测试其与泛素的结合。如果发现这些基序对泛素相互作用很重要，那么不结合泛素的SLX4突变异构体将被测试定位到损伤位点，以及在体外和体内的HJ分解活性。SLX4在端粒酶阴性（ALT）细胞的端粒中被观察到，最近的一项研究强调了SLX4复合物的亚基MUS81在ALT细胞端粒重组中的重要性。在第一个目标的最后一部分，将研究SLX4在ALT细胞端粒募集MUS81中的作用。这将通过siRNA消耗SLX4，结合共沉淀和MUS81与端粒结合蛋白TRF2的荧光定位实验来验证。在第二个目标中，将阐明SLX4解析Holliday结的机理细节。在SLX4复合体中，Holliday连接的对称切割是由SLX1酶催化的，而MUS81-EME1内切酶被认为通过“缺口-反缺口”机制切割hj样结构。一个具有模拟Holliday结的十字形延伸的质粒将被用来研究哪个过程更有利于另一个过程，并首次确定SLX1-SLX4复合物通过同步机制切割HJ的两条链，类似于RuvC等材料HJ分解酶所显示的机制。SLX1和SLX4的基于结构的诱变也将被执行，以检查控制SLX1依赖的HJ分辨率的选择性和对称性的分子因素。在这些体外实验结果之后，将进行实验，测试体内不对称与对称HJ分解活性的作用。

DESCRIPTION (provided by applicant): This training program will be carried out at the post-doctoral level in the laboratory of Dr. Wade Harper at Harvard Medical School. The research study, which is estimated to require 3 years to complete will focus on a recently discovered Holliday junction resolvase component, SLX4. This is an excellent system to study DNA recombinational repair. Numerous technologies exist both in Dr. Harper's lab and at Harvard Medical School which should greatly facilitate the proposed experimental studies such as proteomics and microscopy imaging facilities. In the first aim of the research study, the regulatory mechanisms controlling SLX4 localization and activation and as well as the role of SLX4 in telomere maintenance will be investigated. Since post-translational modifications are important elements in the localization of proteins involved in the damage response, SLX4 and its associated factors will be examined in the context of these processes. Mass spectrometric approaches will be used to identify residues on SLX4 that are phosphorylated in the absence and presence of DNA damage. These residues will be mutated to test if abrogation of SLX4 phosphorylation at specific residues affects its recruitment to sites of damage, or affects its HJ resolution activity. SLX4 contains two tandem ubiquitin interaction motifs and thus will be tested for binding to ubiquitin. If these motifs are found to be important for ubiquitin interaction, SLX4 mutant isoforms that do not bind ubiquitin will be tested for localization to damage sites, as well as HJ resolution activity in vitro and in vivo. SLX4 has been observed at telomeres in telomerase- negative (ALT) cells and a recent study highlights the importance of MUS81, a subunit of the SLX4 complex, in telomere recombination in ALT cells. In the final part of the first aim, the role of SLX4 in the recruitment of MUS81 to telomeres in ALT cells will be examined. This will be tested by depleting SLX4 by siRNA coupled with co-precipitation and fluorescence-based localization experiments of MUS81 with the telomere-binding protein, TRF2. In the second aim, the mechanistic details of SLX4 resolution of Holliday junctions will be elucidated. In SLX4 complexes, symmetrical cleavage across Holliday junctions is catalyzed by the SLX1 enzyme, while the MUS81-EME1 endonuclease is thought to cleave HJ-like structures through a "nick- counternick" mechanism. A plasmid with a cruciform extension that mimics a Holliday junction will be utilized to examine what favors one process over the other, and to establish for the first time that the SLX1-SLX4 complex cleaves both strands of the HJ by a synchronous mechanism, analogous to that displayed by baterial HJ resolvases such as RuvC. structure-based mutagenesis of SLX1 and SLX4 will also be performed to examine the molecular factors that control the selectivity and symmetry of SLX1-dependent HJ resolution. These in vitro results will be followed by experiments that test the role of asymmetric versus symmetric HJ resolvase activity in vivo. PUBLIC HEALTH RELEVANCE: This training program will be devoted to the study of the SLX4 Holliday junction resolvase component, which interacts with multiple elements of recombination-mediated genome maintenance and stability. This will include investigating the recruitment of SLX4 to sites of DNA damage and mechanistic aspects of its catalytic activity. Since SLX4 occupies a central node in homologous recombination, a process that is often deregulated in human cancers, this program can potentially make significant contributions to human health and disease.