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: 8319692
• 负责人: Mohamed Firaz Mohideen
• 金额: $ 5.39万
• 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/8319692
• 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）：该培训计划将在哈佛医学院 Wade Harper 博士的实验室中以博士后水平进行。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霍利迪连接体分解酶成分 SLX4。这是一个研究 DNA 重组修复的优秀系统。哈珀博士的实验室和哈佛医学院都拥有许多技术，这些技术将极大地促进拟议的实验研究，例如蛋白质组学和显微成像设施。 该研究的第一个目标是研究控制 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元素，因此将在这些过程的背景下检查 SLX4 及其相关因子。质谱方法将用于识别 SLX4 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，以测试特定残基处 SLX4 磷酸化的废除是否会影响其招募到损伤位点，或影响其 HJ 解析活性。 SLX4 包含两个串联泛素相互作用基序，因此将测试与泛素的结合。如果发现这些基序对于泛素相互作用很重要，则将测试不结合泛素的 SLX4 突变同种型在损伤位点的定位，以及体外和体内的 HJ 解析活性。 SLX4 已在端粒酶阴性 (ALT) 细胞的端粒上观察到，最近的一项研究强调了 MUS81（SLX4 复合物的一个亚基）在 ALT 细胞端粒重组中的重要性。在第一个目标的最后部分，将检查 SLX4 在将 MUS81 招募到 ALT 细胞端粒中的作用。这将通过 siRNA 耗尽 SLX4，并结合 MUS81 与端粒结合蛋白 TRF2 的共沉淀和基于荧光的定位实验来进行测试。在第二个目标中，将阐明霍利迪连接点的 SLX4 分辨率的机制细节。在 SLX4 复合物中，霍利迪连接处的对称切割由 SLX1 酶催化，而 MUS81-EME1 核酸内切酶被认为通过“切口-反切口”机制切割 HJ 样结构。具有模拟霍利迪连接的十字形延伸的质粒将被用来检查什么有利于一个过程而不是另一个过程，并首次确定SLX1-SLX4复合物通过同步机制切割HJ的两条链，类似于RuvC等细菌HJ解离酶所显示的机制。还将对 SLX1 和 SLX4 进行基于结构的诱变，以检查控制 SLX1 依赖性 HJ 分辨率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，测试体内不对称与对称 HJ 解离酶活性的作用。