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

描述（由申请人提供）：本培训计划将在哈佛医学院Wade哈珀博士的实验室进行博士后水平的培训。这项研究预计需要3年时间才能完成，将重点关注最近发现的Holliday junction resolvase成分SLX 4。这是一个研究DNA重组修复的理想系统。哈珀博士的实验室和哈佛医学院都有许多技术，这些技术应该大大促进拟议的实验研究，如蛋白质组学和显微镜成像设备。 在研究的第一个目的中，将研究控制SLX 4定位和激活的调控机制以及SLX 4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，SLX 4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SLX 4上在不存在和存在DNA损伤的情况下磷酸化的残基。这些残基将被突变以测试在特定残基处SLX 4磷酸化的消除是否影响其向损伤位点的募集或影响其HJ分解活性。SLX 4含有两个串联的泛素相互作用基序，因此将测试其与泛素的结合。如果发现这些基序对于泛素相互作用是重要的，则将测试不结合泛素的SLX 4突变体同种型在损伤位点的定位以及体外和体内的HJ分解活性。已在端粒酶阴性（ALT）细胞中的端粒处观察到SLX 4，并且最近的研究突出了MUS 81（SLX 4复合物的亚基）在ALT细胞中的端粒重组中的重要性。在第一个目标的最后部分，将检查SLX 4在将MUS 81募集到ALT细胞中的端粒中的作用。这将通过siRNA耗尽SLX 4与MUS 81与端粒结合蛋白TRF 2的共沉淀和基于荧光的定位实验来测试。在第二个目标中，将阐明霍利迪路口SLX 4分辨率的机制细节。在SLX 4复合物中，SLX 1酶催化霍利迪连接处的对称切割，而MUS 81-EME 1内切核酸酶被认为通过“切口-反切口”机制切割HJ样结构。将利用具有模拟霍利迪连接的十字形延伸的质粒来检查是什么使一个过程优于另一个过程，并首次建立SLX 1-SLX 4复合物通过同步机制切割HJ的两条链，类似于细菌HJ解离酶如RuvC所显示的机制。还将对SLX 1和SLX 4进行基于结构的诱变，以检查控制SLX 1依赖性HJ拆分的选择性和对称性的分子因素。这些体外结果之后将进行实验，测试不对称与对称HJ解离酶活性在体内的作用。 公共卫生相关性：该培训计划将致力于SLX 4霍利迪连接解离酶组分的研究，该组分与重组介导的基因组维持和稳定性的多个元件相互作用。这将包括研究SLX 4在DNA损伤位点的募集及其催化活性的机制方面。由于SLX 4在同源重组中占据了一个中心节点，这一过程在人类癌症中经常被解除管制，因此该程序可能对人类健康和疾病做出重大贡献。

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.