DNA Triplexes and Replication

DNA 三链体和复制

• 批准号: 9405794
• 负责人: Sergei Mirkin
• 金额: $ 32.57万
• 依托单位: University of Illinois at Chicago
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9405794&HistoricalAwards=false
• 关键词: DNA; Triplexes; Replication

Abstract Mirkin This project focuses on the role of DNA triplexes in DNA replication. We will study homopurine-homopyrimidine sequences with varying extents of mirror symmetry that form intramolecular H-DNA triplexes in vitro. We have recently shown that these sequences cause purified DNA polymerase to terminate in double- or single-stranded DNA templates. Termination sites precisely match triplex junctions defined by chemical probing. Therefore, we suggest that the formation of triplexes prior or during DNA polymerization is responsible for premature termination of DNA synthesis. We have also found that certain orientations and positions of these sequences severely affect the maintenance of bacterial plasmids in vivo. The specific aim of this project is to prove that the formation of triplexes inhibits DNA replication both in vitro and in vivo. We will study the influence of DNA triplexes on DNA synthesis in the reconstituted replication system of bacteriophage T7 and analyze the effects of accessory replication proteins, including single-stranded DNA-binding protein and helicase-primase, on triplex-caused termination. We will characterize the formation of H-like DNA structures in E.coli cells by chemical modification of intracellular DNA followed by sequence level detection of modified bases using Maxam-Gilbert sequencing. We will measure the repression caused by cloned triplex-forming DNA sequences of the replication of the pBluescript plasmid in E.coli cells by assaying the rate of replication by the incorporation of radiolabeled precursors into DNA. We will then visualize the fine pattern of replication fork movement by two-dimensional gel electrophoresis. The long term goal of this work is to reveal the mechanisms of the inhibitory effect of triplexes on DNA synthesis in vitro and to understand the role of these structures in the regulation of DNA replication in vivo. %%% DNA usually exists as a matched pair of long molecules in a structure called double- stranded or duplex DNA. However, particular DNA segments may form an unusual structure where three, rather than two, DNA strands interact with each other. This structure is called triplex DNA. We have found that the purified cellular machinery necessary to duplicate DNA can not efficiently pass through triplex structures within DNA, leading to a block replication. Such an effect could be used to selectively inhibit the multiplication of particular DNAs such as bacteria or viruses. This aims to elucidate the inhibitory effects of triplexes on DNA replication, both in test tubes and in living cells. ***

摘要Mirkin这个项目着重于DNA三心经在DNA复制中的作用。我们将研究具有各种镜子对称性的均尿胺 - 摩pyrimidine序列，这些镜子在体外形成分子内H-DNA三元素。我们最近表明，这些序列引起纯化的DNA聚合酶在双链或单链DNA模板中终止。终止位点恰好匹配由化学探测定义的三重连接。因此，我们建议在DNA聚合之前或在DNA聚合过程中形成三个DNA合成的过早终止。我们还发现，这些序列的某些方向和位置严重影响了体内细菌质粒的维持。该项目的具体目的是证明三元组的形成在体外和体内都抑制了DNA复制。我们将研究噬菌体T7的重构复制系统中DNA三位体对DNA合成的影响，并分析辅助复制蛋白的影响，包括单链的DNA结合蛋白和Heliacase-primase-trimase-primase，对三链的终止终止。我们将通过化学修饰细胞内DNA的化学修饰，然后使用Maxam-Gilbert测序来表征大肠杆菌细胞中H样DNA结构的形成。 我们将通过通过将放射性标记的前体掺入DNA中分析复制速率来衡量由克隆的三型形成的DNA序列在大肠杆菌细胞中复制的复制。然后，我们将通过二维凝胶电泳可视化复制叉运动的精细模式。这项工作的长期目标是揭示三环对DNA合成在体外的抑制作用的机制，并了解这些结构在体内DNA复制中的作用。 %% %% DNA通常以匹配的一对长分子而存在，称为双链或双链DNA。但是，特定的DNA段可能形成一个不寻常的结构，其中三个而不是两个，DNA链相互作用。该结构称为三型DNA。我们发现，复制DNA所需的纯化的细胞机制无法有效地通过DNA内的三元结构，从而导致块复制。这种作用可用于选择性抑制特定DNA（例如细菌或病毒）的繁殖。这旨在阐明在测试管和活细胞中，三元对DNA复制的抑制作用。 ***