SLX4 in Nuclease Recruitment

SLX4 在核酸酶招募中的应用

• 批准号: 9973223
• 负责人: Paula Louise Fischhaber
• 金额: $ 35万
• 依托单位: CALIFORNIA STATE UNIVERSITY NORTHRIDGE
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-08 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9973223
• 关键词: Address; Aging; Area; Biochemical; Biochemistry; Biological; Cancer Etiology; Cell Cycle; Cell division; Cells; Chemical Agents; Chromatin; Chromosome abnormality; Chromosomes; Clinical; Complex; Critical Pathways; Cruciform DNA; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; DNA Sequence; DNA lesion; DNA strand break; Data; Double Strand Break Repair; ERCC1 gene; Elderly; Eukaryota; Event; Excision; Family; Fluorescence Microscopy; Future; G1 Phase; G2 Phase; Genes; Genetic; Genetic Materials; Genome; Genomic Instability; Human; In Vitro; Investigation; Ionizing radiation; Laboratories; Location; Malignant Neoplasms; Methods; Mitosis; Molecular; Monitor; Mutagenesis; Mutation; Pathogenesis; Pathway interactions; Pharmacologic Substance; Phase; Phosphorylation; Play; Prevention therapy; Process; Proteins; Regulation; Reporting; Research; Role; S Phase; Saccharomyces cerevisiae; Signal Transduction; Site; Source; Stretching; Surgical Flaps; Symptoms; Techniques; Time; Ultrafine; Work; Yeasts; cancer prevention; cancer risk; cancer therapy; chromatin immunoprecipitation; endodeoxyribonuclease SceI; endonuclease; experimental study; fluorescence imaging; information model; live cell imaging; new therapeutic target; novel; nuclease; preservation; recruit; repaired; restoration

PROJECT SUMMARY AND ABSTRACT Ionizing radiation and chemical agents induce strand breaks in DNA, which give rise to mutations and chromosomal alterations that can cause cancer. One of the chief biological defenses against DNA strand breaks is Double-Strand Break (DSB) repair, a complicated family of biologic pathways. Some modes of DSB Repair can proceed with full restoration of the DNA sequence, but others result in significant sequence change or loss. Many important questions remain regarding biochemical requirements for pathway selection. Within these broader issues are more specific questions regarding the mechanism of recruitment of proteins that participate in some DSB repair pathways but not others. In baker's yeast (S. cerevisiae), Slx4 protein recruits any of several endonucleases to DSB sites, which either remove an extraneous nonhomologous stretch of DNA or incise a four-way junction of DNA (a Holliday Junction) as one of the last steps of the repair event. The endonucleases recruited by Slx4 include Rad1-Rad10, Mus81-Mms4 and Yen1, but the biochemical details governing the selection of one endonuclease over another are not understood in sufficient detail. It is becoming increasingly clear that the phase of the cell cycle plays a critical role in endonuclease access and engagement of the DSB site. This project will detail the role of Slx4 in recruitment of endonucleases to DSBs in the yeast S. cerevisiae, focusing on why the SLX4 gene is needed for recruitment of endonuclease Rad1-Rad10 in some phases of the cell cycle but not others. The specific aims of this proposal are to: 1) determine whether cells that are not actively engaged in cell division (those in G1) require SLX4 for repair product formation, 2) investigate whether Rad1-Rad10 recruitment to several specific types of DSB sites depends on SLX4 only in S phase, 3) investigate whether checkpoint signal dampening by Slx4 plays a role in SLX4-dependent recruitment of Rad1-Rad10 in DSB repair in dividing cells and, 4) determine whether Rad1-Rad10 colocalizes with Mus81-Mms4 or Yen1 in last-minute DNA repair during the final moments of chromosome separation in cell division and if such localization is SLX4-dependent. These aims will be investigated with a variety of experimental techniques, including a relatively novel fluorescence microscopy approach in which DSBs will be induced and their repair monitored by fluorescence imaging of convergent fluorescent signals. In vitro techniques such as quantitative PCR and Chromatin Immunoprecipitation will also be used to provide corroborating results. These experiments will address important questions regarding the genetic and biochemical requirements for recruitment of Rad1-Rad10, Mus81-Mms4 and Yen1 to DSB sites. All three nucleases are conserved in all eukaryotes including humans. Understanding the molecular basis for genome instability will inform our understanding of the causes of cancer and aging, and will be important for advancing clinical strategies to minimize human suffering.

项目摘要和摘要 电离辐射和化学剂在DNA中诱导链断裂，从而引起突变和 可能导致癌症的染色体改变。针对DNA链的主要生物防御力之一 断裂是双链断裂（DSB）修复，这是一个复杂的生物途径家族。 DSB的一些模式 维修可以进行完全恢复DNA序列，但其他维修会导致显着的序列变化 或损失。关于途径选择的生化要求仍然存在许多重要问题。 在这些更广泛的问题中，关于招募机制的更具体问题 参与某些DSB维修途径的蛋白质，但没有参与其他蛋白质。在贝克的酵母（S. cerevisiae）中，SLX4 蛋白质将几个核酸内切酶中的任何一个招募到DSB位点，它们要么去除无关紧要的 DNA的非同源伸展或开裂的DNA四向连接（霍利迪交界处）是最后一个 维修事件的步骤。 SLX4招募的核核酸酶包括Rad1-RAD10，MUS81-MMS4和Yen1， 但是，尚不理解管理一种核酸内切酶在另一个核酸内切清除酶选择的生化细节。 足够的细节。越来越清楚的是，细胞周期的阶段在 DSB网站的核酸内切酶访问和参与度。该项目将详细介绍SLX4在招聘中的作用 在酵母菌S.酿酒酵母中对DSB的核酸酶，重点介绍为什么需要招募SLX4基因 核酸内切酶rad1-rad10在细胞周期的某些阶段，而不是其他阶段。这个特定的目的 建议是：1）确定未积极参与细胞分裂的细胞（G1中的细胞）是否需要 SLX4用于维修产品的形成，2）调查RAD1-RAD10是否募集到几种特定类型的 DSB位点仅在S阶段取决于SLX4，3）研究检查点信号是否通过SLX4抑制 在分裂细胞中DSB修复中rad1-Rad10的SLX4依赖性募集中起作用，4）确定 RAD1-RAD10是在最后一分钟的DNA修复中与MUS81-MMS4或Yen1共定位 细胞分裂中染色体分离的力矩，如果这种定位是依赖性的。 这些目标将通过各种实验技术进行研究，包括相对新颖 荧光显微镜方法将诱导DSB，并通过荧光监测其修复 收敛荧光信号的成像。体外技术，例如定量PCR和染色质 免疫沉淀也将用于提供佐证的结果。这些实验将解决 关于募集RAD1-RAD10的遗传和生化要求的重要问题， MUS81-MMS4和YEN1到DSB站点。在包括人类在内的所有真核生物中，这三个核酸酶均保守。 了解基因组不稳定性的分子基础将为我们了解我们对癌症原因的理解 和衰老，对于促进临床策略以最大程度地减少人类苦难至关重要。