DNA Replication Initiation Sites in Mammalian Cells

哺乳动物细胞中的 DNA 复制起始位点

• 批准号: 9988576
• 负责人: CARL L SCHILDKRAUT
• 金额: $ 24.35万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-08 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9988576
• 关键词: Architecture; Automobile Driving; Biological; CCCTC-binding factor; Cancer Etiology; Cell Cycle; Cell physiology; Cells; Chromatin Loop; Chromosome Fragile Sites; Chromosome Structures; Chromosomes; Complement; Complex; DNA; DNA Damage; DNA Sequence; DNA biosynthesis; DNA replication fork; Degenerative Disorder; Development; Disease; Elements; Ensure; Etiology; Event; Failure; Fanconi' s Anemia; Functional disorder; G-Quartets; Genetic; Genetic Transcription; Genome; Genome Stability; Genomic Instability; Goals; Human; Hybrids; Lead; Length; Link; Maintenance; Malignant Neoplasms; Mammalian Cell; Mediating; Mus; Nature; Normal Cell; Pathologic; Pathway interactions; Play; Polymerase; Premature aging syndrome; Protein Deficiency; Proteins; RNA; Recovery; Repetitive Sequence; Replication Initiation; Replication Origin; Role; Site; Stress; Structure; Telomere Shortening; Work; age related; base; chromosome replication; genome integrity; genome-wide; high risk; inhibitor/antagonist; initiation site of DNA replication; insight; origin recognition complex; prevent; recruit; repaired; response; single molecule; telomere; telomere loss; tumorigenesis

PROJECT SUMMARY / ABSTRACT Repetitive DNA sequences often challenge DNA replication, which can lead to genomic instability. Aberrant replication of two repeat-rich regions in particular, telomeres and common fragile sites (CFS), have pathological consequences. The goal of this proposal is to elucidate mechanisms that ensure normal telomere and CFS replication and protect genomic integrity. Replication fork stalling at repetitive sequences is thought to be a main causative factor underlying both telomere- and CFS-associated genomic instability. Using our single molecule approach, termed SMARD (single molecule analysis of replicated DNA), we have shown that stalling occurs at both telomeres and CFS. Therefore we propose to examine how stalling is overcome at these important chromosomal elements with the following aims. In Aim1, we will establish the role of replication initiation within telomeres in resuming stalled replication and maintaining proper telomere function. Initiation of replication ahead of a stalled fork is a key replication recovery response. We will disrupt replication fork progression in human and mouse cells using replication inhibitors, G-quadruplex stabilizers and replication barriers, and examine telomere replication to establish that telomeric initiation will occur to rescue stalled replication. Key events driving telomere-associated genetic instability are telomere shortening and failure to suppress DNA damage responses and deleterious repair (telomere dysfunction). We will determine if reduced or compromised telomeric initiation leads to defective telomere replication resulting in telomere loss and dysfunction, to establish whether telomeric initiation provides a protective mechanism for maintaining proper telomere function. In Aim 2, we will establish mechanism(s) that facilitate the replication of stall-prone regions in CFS loci. Our preliminary studies reveal that the Fanconi anemia complementation group D2 protein (FANCD2) plays an important role in facilitating CFS replication by alleviating replication pausing. Thus, we will perform studies to further define its role in CFS replication. Since FANCD2 interacts with translesion polymerases, which have been implicated in CFS replication, we will establish whether FANCD2 recruits these polymerases to aid in CFS replication. We will also investigate whether FANCD2 alleviates transcription- associated obstacles to replication fork progression including RNA: DNA hybrids, because FANCD2–deficient cells display an increased abundance these hybrids genome-wide. Our preliminary studies reveal that changes in replication origin usage are associated with FANCD2 deficiency. Because altered origin usage is linked to changes in chromosome organization, we will determine if FANCD2 promotes a chromosome architecture that facilitates accurate CFS replication. We expect that these proposed studies will both greatly increase our understanding of telomere and CFS replication and allow us to establish new paradigms for replication of repetitive elements and their role in disease etiology.

项目总结/摘要 重复的DNA序列经常挑战DNA复制，这可能导致基因组不稳定。异常 特别是端粒和共同脆性位点（CFS）这两个重复序列丰富的区域的复制， 病理后果。这项提案的目的是阐明确保正常端粒的机制， 和CFS复制并保护基因组完整性。复制分叉在重复序列处停顿被认为是 可能是端粒和CFS相关基因组不稳定性的主要致病因素。使用我们的单曲 分子方法，称为SMARD（复制DNA的单分子分析），我们已经表明， 发生在端粒和CFS上。因此，我们建议研究如何克服这些拖延 重要的染色体元件，具有以下目的。在目标1中，我们将建立复制的角色 端粒内的启动恢复停滞的复制和维持适当的端粒功能。开始 在停滞的分叉之前进行复制是关键的复制恢复响应。我们会破坏复制叉 使用复制抑制剂、G-四链体稳定剂和复制抑制剂在人类和小鼠细胞中的进展 障碍，并检查端粒复制，以建立端粒起始将发生拯救停滞 复制的导致端粒相关遗传不稳定的关键事件是端粒缩短和未能 抑制DNA损伤反应和有害修复（端粒功能障碍）。我们将确定是否减少 或受损的端粒起始导致端粒复制缺陷，导致端粒丢失， 功能障碍，以确定端粒起始是否提供了维持适当的保护机制， 端粒功能在目标2中，我们将建立机制，促进易失速区域的复制 CFS基因座。我们的初步研究表明，范可尼贫血互补组D2蛋白 （FANCD 2）通过减轻复制暂停在促进CFS复制中起重要作用。因此，我们将 开展研究，进一步确定其在推广爱幼学校方面的作用。由于FANCD 2与translesion相互作用， 我们将确定FANCD 2是否招募这些与CFS复制有关的聚合酶， 聚合酶来帮助CFS复制。我们还将研究FANCD 2是否会抑制转录- 与复制叉进展相关的障碍，包括RNA：DNA杂交，因为FANCD 2缺陷 细胞显示出这些杂合体在全基因组范围内的增加的丰度。我们的初步研究显示 在复制起点使用与FANCD 2缺陷有关。因为改变的原产地使用与 染色体组织的变化，我们将确定FANCD 2是否促进染色体结构， 有助于准确复制CFS。我们预计，这些拟议的研究将大大增加我们的 了解端粒和CFS复制，使我们能够建立新的复制模式， 重复元素及其在疾病病因学中的作用。