DNA Triplexes and Replication

米尔金这个项目的重点是DNA三联体在DNA复制中的作用。我们将研究在体外形成分子内H-DNA三联体的具有不同程度镜像对称性的高嘌呤-高嘧啶序列。我们最近发现，这些序列导致纯化的DNA聚合酶终止于双链或单链DNA模板。末端位点与化学探测定义的三链连接精确匹配。因此，我们认为DNA聚合前或聚合过程中形成的三联体是导致DNA合成提前终止的原因。我们还发现，这些序列的某些方向和位置严重影响了细菌质粒在体内的维持。这个项目的具体目的是证明三联体的形成在体外和体内都能抑制DNA复制。我们将研究在重组的T7噬菌体复制系统中DNA三联体对DNA合成的影响，并分析辅助复制蛋白，包括单链DNA结合蛋白和解旋酶-Primase在三联体引起的终止中的作用。我们将通过对细胞内DNA进行化学修饰，然后使用Maxam-Gilbert测序对修饰的碱基进行序列水平检测，来表征大肠杆菌细胞中类H DNA结构的形成。我们将通过将放射性标记的前体掺入DNA来检测克隆的三链形成DNA序列对pBluescrip在大肠杆菌细胞中复制的抑制作用。然后，我们将通过二维凝胶电泳法显示复制叉运动的精细模式。这项工作的长期目标是揭示三联体在体外抑制DNA合成的机制，并了解这些结构在体内DNA复制调控中的作用。DNA通常以一对匹配的长分子存在于一种称为双链或双链DNA的结构中。然而，特定的DNA片段可能会形成一种不寻常的结构，其中三条而不是两条DNA链相互作用。这种结构被称为三链DNA。我们发现，复制DNA所需的纯化细胞机制不能有效地通过DNA内的三链结构，导致块复制。这种效应可以用来选择性地抑制细菌或病毒等特定DNA的增殖。这是为了阐明三联体对DNA复制的抑制作用，包括在试管中和在活细胞中。***

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. ***