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: 10298825
• 负责人: CARL L SCHILDKRAUT
• 金额: $ 53.16万
• 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 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10298825
• 关键词: Adopted; Affect; Artificial Human Chromosomes; Binding; Binding Proteins; Biological; Cancer Etiology; Cell Line; Cell physiology; Cells; Centromere; Chromosome 22; Chromosomes; DNA; DNA Sequence; DNA Structure; DNA biosynthesis; DNA replication fork; DNA-Directed DNA Polymerase; Degenerative Disorder; Dependence; Elements; Ensure; Fiber; Functional disorder; Genetic; Genetic Transcription; Genome; Genomic Segment; Genomics; Goals; Human; Human Chromosomes; Individual; Lead; Light; Link; Maintenance; Mammalian Cell; Mediating; Microscopy; Microtubules; Modeling; Monoubiquitination; Movement; Normal Cell; Pathologic; Play; Polymerase; Process; Repetitive Sequence; Resolution; Ribosomal DNA; Role; Satellite DNA; Site; Stress; Structure; Testing; Time; Variant; Y Chromosome; age related; base; cancer therapy; chromosome replication; druggable target; genome integrity; initiation site of DNA replication; insight; novel; prevent; programs; recruit; response; single molecule; tau Proteins; tau expression; telomere

PROJECT SUMMARY / ABSTRACT Repetitive sequences create difficult-to-replicate (DTR) regions that can stall replication, threatening genomic integrity. Ribosomal DNA (rDNA), centromeric alpha (α) satellites and telomeres are repetitive DTR regions whose sequences form structures that impede replicative DNA Polymerases (Pols). The goal of this project is to gain insights into the factors that affect the accurate replication of these distinct classes of biologically important repetitive elements. We propose to examine factors that can impact their replication and key mechanisms that allow these chromosomal elements to be stably maintained. In Aim 1 we will investigate the role of translesion synthesis (TLS) Pols in replicating rDNA, α-satellite and telomere DTR regions. Evidence implicating TLS Pols in the maintenance of repetitive DTR loci strongly suggests TLS Pols play a role in enabling normal replication of repetitive DTR sequences, likely through replicative Pol – TLS Pol exchange. We will determine the extent of replicative Pol – TLS Pol exchange during replication at the single molecule level in individual live cells using a cutting-edge novel super resolution microscopy approach we have developed. We will determine the dynamics of polymerase exchange at rDNA, α-satellite and telomere loci and establish requirements for exchange including the involvement of PCNA and its monoubiquitination. Our hypothesis that TLS Pols participate in the normal replication of rDNA, α-satellite and telomere regions predicts that these polymerases consequently maintain the stability of these loci. We will test this prediction by determining if reduced TLS Pol activity compromises the stability of these DTR regions in unstressed cells. In Aim 2, we will elucidate the replication programs of the human rDNA and α-satellite repeated sequences and determine features that impact their replication. We will employ naturally occurring repeat loci and ectopically introduced chromosomes carrying defined and uniquely distinguishable human rDNA or centromere α-satellite repeats as model loci to establish specific replication programs. Using these model loci, we will determine the contribution of rDNA sequence/repeat arrangement on their replication program and analyze repeat stability in these loci to establish whether specific sequences/arrangements are more prone to instability. We will also investigate the potential impact of the microtubule binding protein tau on rDNA replication, as its binding to rDNA has been shown to be involved in replication-mediated rDNA instability. Tau also binds to centromeric α- satellite DNA, potentially affecting its replication and stability. Thus, we will determine the effect of tau expression on rDNA and α-satellite replication programs. We expect that these proposed studies will both greatly increase our understanding of rDNA, α-satellite and telomere replication and allow us to establish new paradigms for their replication. Furthermore, with TLS Pols currently being evaluated as druggable targets, particularly in cancer treatment, our studies will provide essential insight on the unanticipated impact of TLS Pol targeting on rDNA, α-satellite and telomere replication and stability.

项目概要/摘要 重复序列会产生难以复制 (DTR) 区域，该区域会阻碍复制，从而威胁基因组 正直。核糖体 DNA (rDNA)、着丝粒 α (α) 卫星和端粒是重复的 DTR 区域 其序列形成阻碍 DNA 聚合酶 (Pols) 复制的结构。该项目的目标是 深入了解影响这些不同类别生物的准确复制的因素 重要的重复元素。我们建议研究可能影响其复制和关键的因素 允许这些染色体元件稳定维持的机制。在目标 1 中，我们将调查 跨损伤合成 (TLS) Pols 在复制 rDNA、α-卫星和端粒 DTR 区域中的作用。证据 暗示 TLS Pols 参与重复 DTR 位点的维持，强烈表明 TLS Pols 在 可能通过复制 Pol – TLS Pol 交换实现重复 DTR 序列的正常复制。 我们将确定单分子复制过程中复制 Pol – TLS Pol 交换的程度 使用尖端的新型超分辨率显微镜方法在单个活细胞中水平 发达。我们将确定 rDNA、α-卫星和端粒位点聚合酶交换的动态， 建立交换要求，包括 PCNA 及其单泛素化的参与。我们的 TLS Pols 参与 rDNA、α-卫星和端粒区域正常复制的假设预测 这些聚合酶因此维持了这些基因座的稳定性。我们将通过以下方式测试这个预测 确定 TLS Pol 活性降低是否会损害无应激细胞中这些 DTR 区域的稳定性。在 目标2，我们将阐明人类rDNA和α-卫星重复序列的复制程序并 确定影响其复制的功能。我们将采用自然发生的重复基因座和异位 引入携带明确且独特可区分的人类 rDNA 或着丝粒 α-卫星的染色体 作为模型基因座重复以建立特定的复制程序。使用这些模型位点，我们将确定 rDNA 序列/重复排列对其复制程序的贡献并分析重复稳定性 这些基因座以确定特定序列/排列是否更容易不稳定。我们还将 研究微管结合蛋白 tau 对 rDNA 复制的潜在影响，因为它与 rDNA 已被证明参与复制介导的 rDNA 不稳定性。 Tau 还结合着丝粒 α- 卫星 DNA，可能影响其复制和稳定性。因此，我们将确定 tau 的作用 rDNA 和 α-卫星复制程序的表达。我们期望这些拟议的研究将 大大增加了我们对 rDNA、α-卫星和端粒复制的理解，使我们能够建立新的 它们的复制范式。此外，目前 TLS Pols 正在被评估为可药物靶标， 特别是在癌症治疗方面，我们的研究将为 TLS 的意外影响提供重要见解 Pol 靶向 rDNA、α-卫星和端粒复制和稳定性。