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: 7099475
• 负责人: ZHIFENG SHAO
• 金额: $ 23.83万
• 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/7099475
• 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）：后生细胞中的DNA复制一直是生物学研究的最重要领域之一。即使在表征参与此过程的蛋白质的表征和监管电路以确保复制的忠诚度方面取得了长足进展，但仍然存在许多根本重要的问题，这些问题尚未完全理解真核基因组中的这些专业区域。其中之一是在大多数后生复制起源的复杂起始区域中选择和维护主动复制起始位点。在这个项目中，我们建议使用两种单分子技术，即原子力显微镜和通过分子梳子进行DNA映射，以直接检查来自单个人类细胞的B-蛋白基因座的DNA复制的细节，作为模型系统。具体而言，我们希望（1）确定在给定的S期间是否可以同时使用B-珠蛋白基因座的起始区的多个起始位点，以确定是否在酵母中发现的机制也应适用于其他较高的Eukaryotes，作为复制起始的一般原则； （2）发现是否可以在随后的S-强度中“记住”一个细胞周期中使用的特定起始位点，以确定是否可以通过表观遗传机制来维持主动的起始位点； （3）为了进一步将这些研究与所选染色体蛋白在这些基因座附近的分布相关联，并在高空间分辨率下对梳子DNA对这些基因座的附近进行了相关性，以确定是否可以将任何已知的染色质结构蛋白直接链接到主动复制起点的选择和维持。通过这些研究，我们不仅希望揭示对复杂复制起源的性质和实用性的关键见解，而且还希望进一步建立这些单个分子方法，以研究单个真核细胞中DNA复制的研究。当将这种方法与其他已建立的技术结合使用时，我们将能够进一步揭示各种分子决定因素和基本复制的基本原理，这些基因组复制也应该具有实际的影响。