DNA Triplexes and Replication

本项目主要研究DNA三联体在DNA复制中的作用。我们将研究具有不同程度镜像对称的同嘌呤-同嘧啶序列，在体外形成分子内H-DNA三联体。我们最近表明，这些序列导致纯化的DNA聚合酶终止在双链或单链DNA模板。终止位点精确匹配由化学探针定义的三重结。因此，我们认为在DNA聚合之前或过程中形成三联体是导致DNA合成过早终止的原因。我们还发现，这些序列的某些取向和位置严重影响细菌质粒在体内的维持。该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。我们将研究重组噬菌体T7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结合蛋白和解旋酶引物酶）对三联体终止的影响。我们将通过对细胞内DNA进行化学修饰，然后使用Maxam-Gilbert测序对修饰碱基进行序列水平检测，来表征大肠杆菌细胞中h样DNA结构的形成。我们将通过将放射性标记的前体结合到DNA中来测定复制速率，从而测量克隆的三联体形成DNA序列对pBluescript质粒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联体对体外DNA合成抑制作用的机制，并了解这些结构在体内DNA复制调节中的作用。DNA通常以一对匹配的长分子形式存在，其结构称为双链或双链DNA。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。这种结构被称为三联体DNA。我们发现复制DNA所必需的纯化细胞机制不能有效地通过DNA内的三重结构，导致阻滞复制。这种效应可用于选择性地抑制特定dna（如细菌或病毒）的增殖。本研究旨在阐明三联体在试管和活细胞中对DNA复制的抑制作用。＊＊＊

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. ***