Single Molecule Studies of Replication Origin in Metazoa

后生动物复制起源的单分子研究

• 批准号: 6772137
• 负责人: ZHIFENG SHAO
• 金额: $ 24.33万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6772137
• 关键词: DNA replication origin; antibody; atomic force microscopy; cell line; chromatin immunoprecipitation; clone cells; eukaryote; genetic mapping; globin; heterochromatin; histones; molecular genetics; nucleic acid purification; nucleic acid structure

DESCRIPTION (provided by applicant): DNA replication in metazoan cells has been one of the most important areas of research in biology. Even though great progress has been made in characterizing the proteins involved in this process and the regulatory circuitry evolved to ensure the fidelity of replication, there are still many fundamentally important questions that have not yet been fully understood about these specialized regions in the eukaryotic genome. One of these is the selection and maintenance of active replication start sites in complex initiation zones found at most metazoan replication origins. In this project, we propose to apply two single molecule techniques, atomic force microscopy and DNA mapping with molecular combing, to directly examine the details of DNA replication at the B-globin locus from individual human cells as a model system. Specifically, we wish (1) to determine whether multiple start sites in the initiation zone at the B-globin locus could be utilized simultaneously in a given S-phase, thus, to determine if a mechanism similar to "origin interference" found in yeast should also apply to other higher eukaryotes as a general principle of replication initiation; (2) to discover whether a specific start site used in one cell cycle could be "memorized" in subsequent S-phases, thus, to determine if an active start site could be maintained by an epigenetic mechanism; (3) to further correlate these studies with the distribution of selected chromosomal proteins in the vicinity of these loci using ChIP and optical mapping on combed DNA at high spatial resolution, thus, to determine if any of the known chromatin structural proteins can be directly linked to the selection and maintenance of active replication start sites. With these studies, not only do we wish to reveal critical insights on the nature and utility of complex replication origins, but also to further establish these single molecule methods for the study of DNA replication in individual eukaryotic cells. When such methods are combined with other established techniques, we will be able to further unravel various molecular determinants and the fundamental principles of genomic replication that should also have practical implications.

描述（由申请人提供）：后生动物细胞中的 DNA 复制一直是生物学最重要的研究领域之一。尽管在表征这一过程中涉及的蛋白质方面已经取得了巨大进展，并且调控电路也得到了进化以确保复制的保真度，但对于真核基因组中的这些特殊区域，仍然存在许多根本上重要的问题尚未完全了解。其中之一是在大多数后生动物复制起点发现的复杂起始区中选择和维护主动复制起始位点。在这个项目中，我们建议应用两种单分子技术，即原子力显微镜和分子梳理DNA作图，以作为模型系统直接检查来自单个人类细胞的B珠蛋白基因座的DNA复制细节。具体来说，我们希望（1）确定B球蛋白基因座起始区的多个起始位点是否可以在给定的S期中同时利用，从而确定在酵母中发现的类似于“起源干扰”的机制是否也应该适用于其他高等真核生物作为复制起始的一般原则； (2) 发现一个细胞周期中使用的特定起始位点是否可以在随后的S期中被“记忆”，从而确定是否可以通过表观遗传机制维持一个活跃的起始位点； (3) 使用 ChIP 和高空间分辨率的梳状 DNA 光学绘图，进一步将这些研究与这些位点附近选定的染色体蛋白的分布联系起来，从而确定是否有任何已知的染色质结构蛋白可以直接与活性复制起始位点的选择和维持相关。通过这些研究，我们不仅希望揭示对复杂复制起点的性质和实用性的重要见解，而且还希望进一步建立这些单分子方法来研究单个真核细胞中的 DNA 复制。当这些方法与其他已建立的技术相结合时，我们将能够进一步阐明各种分子决定因素和基因组复制的基本原理，这也应该具有实际意义。