Assay for Detection of Homologous DNA Interactions

同源 DNA 相互作用的检测分析

• 批准号: 10614927
• 负责人: Valentin Boerner
• 金额: $ 47.73万
• 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/10614927
• 关键词: Adsorption; Alleles; 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 Diseases; Genetic Recombination; Genome; Genome Mappings; Genome Stability; Genome engineering; Genomic Instability; Genomic Segment; Germ Lines; Glean; Goals; Haploidy; 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; chromosome conformation capture; chromosome missegregation; detection assay; ds-DNA; genome-wide; homologous recombination; improved; insight; model organism; 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忠实的染色体 分离和有效的染色体断裂修复。在生殖系中，配对在 同源染色体是减数分裂过程中遗传交换的前提，确保 同源染色体的分离和新的等位基因组合的创造。中的缺陷 同源DNA配对导致染色体错误分离和总染色体 重排、癌症相关疾病、过早衰老和出生缺陷。 此外，单等位基因表达的等位基因选择也被认为涉及瞬时 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染色体中的作用 功能。我们假设配对优先发生在基因决定的 染色体区域由环分隔，其中配对较低或不存在。我们的调查 致力于开发全基因组检测同源DNA的方法 芽殖酵母作为模式生物的相互作用。我们已经开发出了相应的 配对捕获(HPC)分析系统，允许识别同源DNA相互作用 在完整的细胞中，在全基因组范围内和核苷酸分辨率上。在我们的具体目标1中，我们 将建立一个检测和量化DNA配对相互作用的原型 减数分裂过程中最大配对的酵母染色体。我们将进一步绘制首选地图 同源染色体之间的关联。在我们的具体目标2中，我们将发展 定量区分相似和相似之间配对相互作用的方法 在相同的DNA片段之间。这将使我们能够区分配对交互 同源染色体之间的，而不是姐妹染色单体之间的。至 提供独立的分析验证，我们将应用HPC进行分支重组 应该与已知的基因交换位置相关的中间体。身份识别 通过HPC分析的首选配对序列将为机械性 对这个无处不在的生物过程的理解。