SLX4 in Nuclease Recruitment

SLX4 在核酸酶招募中的应用

• 批准号: 10225579
• 负责人: Paula Louise Fischhaber
• 金额: $ 36.25万
• 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 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10225579
• 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.

项目总结和摘要 电离辐射和化学制剂会引起DNA链断裂，从而引起突变， 可能导致癌症的染色体变异对抗DNA链的主要生物防御之一 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 修复可以通过DNA序列的完全恢复来进行，但其他修复会导致显著的序列变化 或损失。许多重要的问题仍然关于途径选择的生化要求。 在这些更广泛的问题中，有一些更具体的问题，涉及征聘机制， 参与某些DSB修复途径但不参与其他途径的蛋白质。在面包酵母（S.酿酒酵母），Slx 4 蛋白质招募任何几种内切核酸酶到DSB位点，这要么去除外来的 DNA的非同源延伸或切割DNA的四向连接（霍利迪连接）作为最后一个 修复事件的步骤。Slx 4募集的核酸内切酶包括Rad 1-Rad 10、Mus 81-Mms 4和Yen 1， 但是控制一种内切核酸酶选择另一种内切核酸酶的生物化学细节在 足够的细节。越来越清楚的是，细胞周期的阶段在细胞周期中起着关键作用。 在一些实施方案中，DSB位点可通过核酸内切酶进入和DSB位点的接合来调节。该项目将详细介绍Slx 4在招募 内切酶与酵母S.酿酒酵母，重点是为什么SLX 4基因是需要招聘 核酸内切酶Rad 1-Rad 10在细胞周期的某些阶段，但不是其他人。具体目标是 建议是：1）确定不积极参与细胞分裂的细胞（G1中的细胞）是否需要 SLX 4用于修复产物形成，2）研究Rad 1-Rad 10是否募集到几种特定类型的细胞中， DSB位点仅在S期依赖于SLX 4，3）调查检查点信号是否被Slx 4抑制 在分裂细胞的DSB修复中，在Rad 1-Rad 10的SLX 4依赖性募集中起作用，4）确定 Rad 1-Rad 10是否与Mus 81-Mms 4或Yen 1在最后一分钟的DNA修复中共定位 细胞分裂中染色体分离的时刻以及这种定位是否是SLX 4依赖性的。 这些目标将与各种实验技术，包括一个相对新颖的调查 荧光显微镜方法，其中DSB将被诱导并通过荧光监测其修复 会聚荧光信号的成像。体外技术，如定量PCR和染色质 免疫沉淀也将用于提供确证结果。这些实验将解决 关于招募Rad 1-Rad 10的遗传和生化要求的重要问题， Mus 81-Mms 4和Yen 1至DSB位点。所有这三种核酸酶在包括人类在内的所有真核生物中都是保守的。 了解基因组不稳定性的分子基础将有助于我们了解癌症的原因 和老化，并将是重要的推进临床策略，以尽量减少人类的痛苦。