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.

项目概要和摘要 电离辐射和化学试剂会引起 DNA 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 链的主要生物防御之一 断裂是双链断裂（DSB）修复，是一个复杂的生物途径家族。 DSB的一些模式 修复可以完全恢复 DNA 序列，但其他修复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，还有一些关于招募机制的更具体的问题。 参与某些 DSB 修复途径但不参与其他途径的蛋白质。面包酵母（酿酒酵母）中，Slx4 蛋白质将几种核酸内切酶中的任何一种招募到 DSB 位点，这些酶要么去除无关的核酸 DNA 的非同源片段或切割 DNA 的四路连接（霍利迪连接）作为最后的连接之一 修复事件的步骤。 Slx4 招募的核酸内切酶包括 Rad1-Rad10、Mus81-Mms4 和 Yen1， 但控制一种核酸内切酶相对于另一种核酸内切酶的选择的生化细节尚不清楚 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至关重要的作用。 DSB 位点的核酸内切酶访问和参与。该项目将详细介绍 Slx4 在招募 酿酒酵母中 DSB 的核酸内切酶，重点关注为何需要 SLX4 基因进行招募 核酸内切酶 Rad1-Rad10 在细胞周期的某些阶段存在，但在其他阶段则不然。本次活动的具体目标 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 SLX4 用于修复产物形成，2) 研究 Rad1-Rad10 是否招募到几种特定类型的 DSB 位点仅在 S 期依赖于 SLX4，3) 研究 Slx4 是否抑制检查点信号 在分裂细胞 DSB 修复中 SLX4 依赖的 Rad1-Rad10 募集发挥作用，4) 确定 Rad1-Rad10 是否在最后一刻的 DNA 修复中与 Mus81-Mms4 或 Yen1 共定位 细胞分裂中染色体分离的时刻以及这种定位是否依赖于 SLX4。 这些目标将通过各种实验技术进行研究，包括相对新颖的 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，例如定量 PCR 和染色质 免疫沉淀也将用于提供确凿的结果。这些实验将解决 关于招募 Rad1-Rad10 的遗传和生化要求的重要问题， Mus81-Mms4 和 Yen1 到 DSB 站点。所有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 和衰老，对于推进减少人类痛苦的临床策略非常重要。