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

人类霍利迪连接解离酶成分 SLX4 在 DNA 重组中的作用

• 批准号: 8521188
• 负责人: Mohamed Firaz Mohideen
• 金额: $ 5.57万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8521188
• 关键词: Active Sites; Affect; Binding; Biological Assay; Boxing; Cell Cycle; Cells; Cleaved cell; Complex; Coupled; Cruciform DNA; DNA; DNA Binding; DNA Damage; DNA Repair; DNA Repair Pathway; DNA damage checkpoint; Data; Deoxyribonuclease I; Disease; Dissociation; Elements; Enzymatic Biochemistry; Enzymes; Fluorescence; Genetic Recombination; Genome; Health; Holliday Junction Resolvases; Human; Image; In Vitro; Indium; Laboratories; Lasers; Link; Maintenance; Malignant Neoplasms; Mediating; Microscopy; Mismatch Repair; Molecular; Mutagenesis; Mutate; Nature; PLK1 gene; Pathway interactions; Phosphorylation; Phosphoserine Motif; Phosphotransferases; Plasmids; Play; Post-Translational Protein Processing; Postdoctoral Fellow; Precipitation; Process; Property; Protein Binding; Protein Isoforms; Proteins; Proteomics; Recruitment Activity; Regulation; Resolution; Resolvase; Role; Scaffolding Protein; Side; Site; Small Interfering RNA; Structure; System; Technology; Telomerase; Telomere Maintenance; Telomere Recombination; Telomere-Binding Proteins; Testing; Time; Training Programs; Ubiquitin; Ubiquitin-Protein Ligase Complexes; Work; base; endonuclease; helicase; homologous recombination; in vitro Assay; in vitro activity; in vivo; irradiation; medical schools; mutant; programs; recombinational repair; repaired; research study; response; telomere; tool; ubiquitin-protein ligase

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.

描述(由申请人提供)：该培训项目将在哈佛医学院韦德·哈珀博士的实验室进行博士后阶段的培训。这项预计需要3年时间完成的研究将集中在最近发现的Holliday连接解析酶成分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，这些技术应该会极大地促进拟议的实验研究，如蛋白质组学和显微成像设施。在这项研究的第一个目标中，将研究控制SLX4定位和激活的调节机制以及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，因此将在这些过程的背景下研究SLX4及其相关因素。质谱学方法将被用来鉴定在没有DNA损伤和存在DNA损伤的情况下，SLX4上被磷酸化的残基。这些残基将被突变，以测试特定残基上SLX4磷酸化的取消是否会影响其在损伤部位的募集，或影响其HJ拆分活性。SLX4包含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情况。如果这些基序被发现对泛素相互作用是重要的，则将测试不结合泛素的SLX4突变异构体在损伤部位的定位以及体外和体内的HJ拆分活性。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复合体的一个亚单位MUS81在ALT细胞端粒重组中的重要性。在第一个目标的最后部分，将研究SLX4在ALT细胞中MUS81招募到端粒中的作用。这将通过siRNA耗尽SLX4，以及MUS81与端粒结合蛋白TRF2的共沉淀和荧光定位实验来验证。在第二个目标中，我们将阐明Holliday结的SLX4分辨的机制细节。在SLX4复合体中，SLX1酶催化跨越Holliday连接的对称切割，而MUS81-EME1内切酶被认为通过“Nick-Counternick”机制切割HJ样结构。一个具有模拟Holliday连接的十字形延伸的质粒将被用来研究什么有利于一个过程而不是另一个过程，并首次确定SLX1-SLX4复合体通过一种同步机制切割HJ的两条链，类似于RuvC等两种HJ解析酶所显示的机制。还将进行SLX1和SLX4的基于结构的突变，以检查控制SLX1依赖的HJ拆分的选择性和对称性的分子因素。在这些体外结果之后，将进行实验，以测试体内不对称和对称HJ解旋酶活性的作用。