DNA REPLICATION INITIATION SITES IN MAMMALIAN CE

我们的长期目标包括阐明几个基本的和长期的 哺乳动物染色体复制的悬而未决的问题。其中包括：1) 是否存在基因定义的序列(复制子)来决定位置 用于启动DNA复制，而不管是否启动 发生在特定地点或非本地化区域？2)什么是 在特定时间暂停复制分叉的意义和机制 站点？3)中是否存在特定于站点终止复制的角色 哺乳动物的染色体？是什么决定了精确的时间复制 S期的特定染色体座方案？ 我们最近观察到潜伏的爱泼斯坦-巴尔 病毒(EBV)的复制在几个方面类似于染色体 在人类细胞中复制。EBV是一种致癌的人类疱疹病毒，它可以 引起传染性单核细胞增多症，与非洲密切相关 Burkitt淋巴瘤和鼻咽癌是最常见的肿瘤之一 亚洲的各种癌症。在感染艾滋病毒的个人或在 EBV相关的免疫抑制移植患者 淋巴增生性疾病可能高发。EBV基因组 在少数B细胞中保持染色体外潜伏状态 大多数人类中的淋巴细胞，并在整个生命周期中保持不变 一辈子。EB病毒表体被宿主细胞机制精确地复制 每个细胞周期一次。对EBV致病机制的认识 复制和维护应该为这方面提供有价值的见解 重要的人类病原体。在本提案中，我们将使用EBV作为模型 回答有关哺乳动物DNA的几个基本问题的系统 复制。具体目标包括：1)我们将确定潜在的身份证件- 建立离域起始区的作用序列，我们 最近在EBV上描述了。为了实现这一点，我们将首先生成 具有离域起始突变的重组EBV基因组 区域。然后，我们将通过两种方法检测这些改良的EBV毒株- 用双向凝胶电泳法鉴定重要的基因序列 离域启动。2)我们将确定顺式和反式作用 EBV基因组中复制分叉暂停的要求。我们 将首先确定叉子被搁置的准确位置，然后 特异性地诱变DNA序列以识别关键元件 必填项。

Our long term goals include elucidating several fundamental and long standing questions in mammalian chromosome replication. These include: 1) Are there genetically defined sequences (replicators) that determine sites for initiation of DNA replication irrespective of whether initiation occurs at specific sites or in delocalized regions? 2) What is the significance and mechanism of pausing of replication forks at specific sites? 3) Is there a role for site-specific termination of replication in mammalian chromosomes? 4) What determines the precise temporal replication program of specific chromosomal loci during the S phase? We have recently made the important observation that latent Epstein-Barr virus (EBV) replication resembles, in several ways, chromosomal replication in human cells. EBV is an oncogenic human herpesvirus that can cause infectious mononucleosis and is closely associated with African Burkitt's lymphoma and nasopharyngeal carcinoma, one of the most common forms of cancer in Asia. In individuals infected with HIV or in immunosuppressed transplantation patients, EBV-associated lymphoproliferative disorders can occur at high frequency. The EBV genome persists extrachromosomally in a latent state in a small number of B lymphocytes in most humans and is maintained throughout their entire lifetime. EBV episomes are duplicated by the host cell machinery precisely once per cell cycle. An understanding of the mechanisms by which EBV replicates and is maintained should provide valuable insights into this important human pathogen. In this proposal we will use EBV as a model system to answer several fundamental questions about mammalian DNA replication. Specific aims include: 1) We will identify potential cis- acting sequences that establish the delocalized initiation region that we have recently described in EBV. To accomplish this we will first generate recombinant EBV genomes with mutations in the delocalized initiation region. We will then examine these modified EBV strains by two- dimensional gel electrophoresis to identify sequences important for delocalized initiation. 2) We will determine the cis- and trans-acting requirements for the pausing of replication forks in the EBV genome. We will first identify the precise location where forks are stalled and then specifically mutagenize DNA sequences to identify the critical elements required.