Single Molecule Studies of Replication Origin in Metazoa

描述（申请人提供）：后生动物细胞中的DNA复制一直是生物学中最重要的研究领域之一。尽管在描述参与这一过程的蛋白质和确保复制保真度的调控回路方面已经取得了很大的进展，但关于真核生物基因组中的这些特殊区域，仍有许多根本性的重要问题尚未得到充分理解。其中之一是在大多数后生动物复制起始点发现的复杂起始区中主动复制起始位点的选择和维护。在本项目中，我们建议应用原子力显微镜和DNA定位与分子梳理两种单分子技术，直接研究个体人类细胞作为模型系统的b -珠蛋白位点DNA复制的细节。具体来说，我们希望(1)确定b -珠蛋白位点起始区的多个起始位点是否可以在给定的s期同时被利用，从而确定类似于酵母中发现的“起源干扰”机制是否也适用于其他高等真核生物，作为复制起始的一般原则；(2)发现在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”，从而确定活性起始位点是否可以通过表观遗传机制维持；(3)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，使用ChIP和高空间分辨率的精梳DNA光学定位，从而确定是否有任何已知的染色质结构蛋白可以直接与主动复制起始位点的选择和维持联系起来。通过这些研究，我们不仅希望揭示复杂复制起源的本质和效用的关键见解，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，我们将能够进一步揭示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。

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.