DNA REPLICATION INITIATION SITES IN MAMMALIAN CE

哺乳动物 CE 中的 DNA 复制起始位点

• 批准号: 2634694
• 负责人: CARL L SCHILDKRAUT
• 金额: $ 40.2万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-01-08 至 1999-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2634694
• 关键词: DNA replication; DNA replication origin; Epstein Barr virus; gel electrophoresis; gene mutation; genetic regulatory element; polymerase chain reaction; recombinant DNA; tissue /cell culture; transcription factor; virus genetics; virus replication

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.

我们的长期目标包括阐明几个基本和长期的 哺乳动物染色体复制的长期问题。这些措施包括：1） 是否有基因定义的序列（复制因子）决定位点 启动DNA复制，无论启动 发生在特定的位置还是在非定域区域？2)是什么 复制叉在特定时间暂停的意义和机制 网站？3)中的特定于站点的复制终止是否有作用 哺乳动物染色体4)是什么决定了精确的时间复制 在S期的特定染色体位点的程序？ 我们最近发现潜伏的爱泼斯坦-巴尔病毒 病毒（EBV）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， 引起传染性单核细胞增多症，并与非洲 伯基特淋巴瘤和鼻咽癌，其中最常见的 癌症在亚洲在感染艾滋病毒的人或 免疫抑制移植患者，EBV相关 淋巴组织增生性疾病可以高频率发生。 EBV基因组 在少量B中以潜伏状态在染色体外持续存在 大多数人的淋巴细胞并在其整个过程中维持 辈子EBV游离体被宿主细胞机器精确复制 每个细胞周期一次。 了解EBV感染的机制 复制和维护应该对此提供有价值的见解 重要的人类病原体 在本提案中，我们将使用EBV作为模型 系统来回答有关哺乳动物DNA的几个基本问题 复制的 具体目标包括：1）我们将确定潜在的顺- 作用序列建立了我们所认为的离域起始区 最近在EB病毒中被描述。为了实现这一点，我们将首先生成 在离域起始处具有突变的重组EBV基因组 地区 然后我们将通过两种方法检查这些修饰的EBV毒株- 三维凝胶电泳，以确定重要的序列 离域引发2)我们将确定顺式和反式作用 在EBV基因组中暂停复制叉的要求。我们 将首先确定叉停止的精确位置，然后 特异性地诱变DNA序列以识别关键元件 必需的.