Single Molecule Studies of Replication Origin in Metazoa

描述(申请人提供)：后生动物细胞中的DNA复制一直是生物学研究中最重要的领域之一。尽管在鉴定参与这一过程的蛋白质和进化调控回路以确保复制的保真度方面已经取得了很大进展，但关于真核基因组中这些专门化区域仍有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始点发现的复杂起始区中选择和维护活跃的复制起始点。在这个项目中，我们建议应用两种单分子技术，原子力显微镜和分子梳理的DNA作图，以单个人类细胞为模型系统，直接检测B-珠蛋白基因座DNA复制的细节。具体地说，我们希望(1)确定在给定的S时相中，是否可以同时利用位于B-珠蛋白基因座起始区的多个起始点，从而确定在酵母中发现的类似于“起源干扰”的机制是否也应该作为复制起始的一般原理适用于其他高等真核生物；(2)发现在一个细胞周期中使用的特定起始点是否可以在随后的S相中被“记忆”，从而确定活跃的起始点是否可以通过表观遗传机制来维持；(3)利用高空间分辨率的芯片和光学作图技术，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点的选择和维持相关联。通过这些研究，我们不仅希望揭示复杂复制起始点的性质和用途，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，我们将能够进一步解开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.