MUTATIONAL ANALYSIS OF E COLI DNA TOPOISOMERASE II

描述：dna拓扑异构酶是改变连接数的酶。 并负责拓扑的内部和内部转换 存在于DNA分子中。这些转换包括否定的 超级线圈，去除负极线圈，打结， 以及DNA的连结和消解。这些酶是已知的 细菌细胞中抗生素和抗肿瘤药物的靶点 细胞。拓扑异构酶可以分为两个不同的类别。类型1 酶通过使DNA螺旋的一条链瞬间断裂来发挥作用， 将单链或双链DNA穿过断口，并重新密封 休息一下。2型酶的工作方式类似，只是它们能使 螺旋的两条链都有一过性断裂。这两种类型的示例 酶在真核生物和原核生物中都存在。DNA拓扑异构酶III (TOPO III)是从大肠杆菌中分离出来的一种1型酶。 该酶的晶体结构已经确定，并且是唯一的 具有活性的拓扑异构酶的结构。这种拓扑异构酶是 拓扑异构酶的独特之处在于它既能作用于DNA，也能作用于RNA 底物。此外，这种酶的同源物已经被发现是 由混杂的、广泛宿主范围的质粒基因组编码，以及 酵母和人类基因组。到目前为止，大肠杆菌是唯一与Topo III类似的 既适合生化分析又适合遗传分析的活动。这个 这笔赠款的具体目标是：1)评估类型1的拟议模型 拓扑异构酶介导的松弛和衰变；2)进一步鉴定 参与底物结合的Topo III多肽的临界残基 和DNA裂解的体外诱变，并试图阐明 分子水平的酶机制；3)尝试鉴定细胞 与Topo III相互作用并绘制Topo III区域的蛋白质 4)抑制topB的表达，并分析其对基因表达的影响 其他拓扑异构酶基因的表达及其在细胞中的状态 细胞内DNA。

DESCRIPTION: DNA topoisomerases are enzymes that alter the linking number of DNA and are responsible for the topological inter- and intraconversions that occur in DNA molecules. These conversions include the negative supercoiling of, the removal of negative supercoils from, the knotting of, and the catenation and decatenation of DNA. These enzyme are the known targets for antibiotics in bacterial cells and anti-tumor drugs in human cells. Topoisomerases can be classified into two distinct groups. Type 1 enzymes act by making a transient break in one strand of the DNA helix, passing a single or double strand DNA through the break, and resealing the break. Type 2 enzymes work in a similar manner except that they make a transient break in both strands of the helix. Examples of both types of enzymes exist in both eucaryotes and prokaryotes. DNA topoisomerase III (Topo III) is a type 1 enzyme that has been isolated from Escherichia coli. The crystal structure of the enzyme has been determined and is the only structure available of an active topoisomerase. This topoisomerase is unique among the topoisomerases in that it can act on both DNA and RNA substrates. In addition, homologues of this enzyme have been found to be encoded by promiscuous, broad host range plasmid genomes as well as the yeast and human genome. Thus far, E. coli is the only Topo III-like activity that is amenable to both biochemical and genetic analysis. The specific aims of this grant are: 1) assess the proposed model for type 1 topoisomerase-mediated relaxation and decatenation; 2) further identify the critical residues of the Topo III polypeptide involved in substrate binding and DNA cleavage using in vitro mutagenesis and attempt to elucidate the enzymatic mechanism at the molecular level; 3) attempt to identify cellular proteins that interact with Topo III and map the regions of Topo III with which they interact; 4) Suppress topB expression and analyze the effect on the expression of other topoisomerase genes and the state of the intracellular DNA.