Assay for Detection of Homologous DNA Interactions

同源 DNA 相互作用的检测分析

• 批准号: 10366921
• 负责人: Valentin Boerner
• 金额: $ 49.46万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10366921
• 关键词: Adsorption; Alleles; Animal Model; Biological Assay; Biological Process; Cell Aging; Cells; Chromatin; Chromosomal Breaks; Chromosome Segregation; Chromosome Structures; Chromosomes; Congenital Abnormality; Cruciform DNA; Cytology; DNA; DNA Ligation; DNA Sequence; Defect; Development; Diagnosis; Digestion; Disease; Elements; Engineering; Ensure; Excision; Failure; Foundations; Frequencies; Gene Dosage; Gene Expression; Genetic; Genetic Recombination; Genome; Genome Mappings; Genome Stability; Genome engineering; Genomic Instability; Germ Lines; Glean; Goals; Head; Health; Hi-C; Homologous Gene; Investigation; Ligation; Link; Malignant Neoplasms; Maps; Mediating; Meiosis; Mitotic; Molecular; Monitor; Nucleotides; Pathologic; Positioning Attribute; Premature Birth; Premature aging syndrome; Proteins; Refractory; Reporter; Resolution; Ribosomal DNA; Role; Saccharomycetales; Sexual Reproduction; Sister Chromatid; Site; Somatic Cell; Specificity; Surface; System; Trans-Activators; Validation; Work; Yeasts; base; chromosome conformation capture; chromosome missegregation; detection assay; ds-DNA; genome-wide; homologous recombination; improved; insight; novel; prevent; prototype; repaired; reproductive; segregation; tool; whole genome; yeast genome

Project Summary Homologous DNA interactions are a key determinant of chromosome structure and genome function. In mitotic cells, pairing between sister chromatids ensures faithful chromosome segregation and efficient chromosome break repair. In the germ line, pairing between homologous chromosomes is a precondition for genetic exchange during meiosis, ensuring segregation of homologous chromosomes and creation of novel allele combinations. Defects in homologous DNA pairing contribute to chromosome missegregation and gross chromosome rearrangements, conditions associated with cancer, premature aging and birth defects. Moreover, allele choice for monoallelic gene expression is also thought to involve transient interactions between homologous DNA segments. Our long-term goal is to understand the molecular mechanism of homologous pairing and its role in chromosome structure and function. We hypothesize that pairing preferentially occurs in genetically determined chromosome regions separated by loops where pairing is low or absent. Our investigation focuses on the development of a genome-wide assay for detection of homologous DNA interactions in budding yeast as a model organism. We have developed the Homologous Pairing Capture (HPC) assay system that allows identification of homologous DNA interactions in intact cells, on a genome-wide scale and at nucleotide resolution. In our Specific Aim 1, we will establish a prototype for detecting and quantitating DNA pairing interactions along maximally paired yeast chromosomes during meiosis. We will further map preferred associations between homologous chromosomes. In our Specific Aim 2, we will develop approaches to quantitatively distinguish pairing interactions between similar from those between identical DNA segments. This will enable us to distinguish pairing interactions between homologous chromosomes from those occurring between sister chromatids. To provide independent assay validation, we will apply HPC to branched recombination intermediates that should correlate with known positions of genetic exchange. Identification of preferred pairing sequences via the HPC assay will lay the foundation for a mechanistic understanding of this ubiquitous biological process.

项目摘要 同源DNA相互作用是染色体结构和基因组的关键决定因素 功能在有丝分裂细胞中，姐妹染色单体之间的配对确保了染色体的忠实性。 分离和有效的染色体断裂修复。在生殖细胞系中， 同源染色体是减数分裂期间遗传交换的先决条件， 同源染色体的分离和新等位基因组合的产生。缺陷 同源DNA配对有助于染色体错误分离和粗染色体 基因重排、与癌症、早衰和出生缺陷相关的疾病。 此外，单等位基因表达的等位基因选择也被认为涉及短暂的 同源DNA片段之间的相互作用。我们的长期目标是了解 同源配对的分子机制及其在染色体结构和 功能我们假设，配对优先发生在基因决定的 被环分开的染色体区域，在那里配对低或不存在。我们的调查 专注于开发用于检测同源DNA的全基因组分析 作为模式生物的芽殖酵母中的相互作用。我们已经开发了Homeland 配对捕获（HPC）分析系统，可识别同源DNA相互作用 在完整的细胞中，在全基因组范围内，在核苷酸分辨率上。在具体目标1中，我们 将建立一个原型，用于检测和定量DNA配对相互作用沿着 最大配对酵母染色体在减数分裂。我们将进一步地图首选 同源染色体之间的联系。在我们的具体目标2中，我们将开发 方法来定量区分配对之间的相互作用相似， 完全相同的DNA片段这将使我们能够区分配对相互作用 同源染色体与姐妹染色单体之间的同源染色体之间的同源染色体。到 提供独立的检测验证，我们将HPC应用于分支重组 这些中间体应该与已知的遗传交换位置相关。鉴定 通过HPC测定优选的配对序列将为 了解这个无处不在的生物过程。