MUTATIONAL ANALYSIS OF E COLI DNA TOPOISOMERASE II

描述：DNA拓扑异构酶是改变DNA连接数的酶。 负责DNA的拓扑间和内转换 发生在DNA分子中。 这些转换包括负 超螺旋，负超螺旋的去除，打结， 以及DNA的连锁和去连锁。 这些酶是已知的 抗生素在细菌细胞中的靶点和抗肿瘤药物在人体中的靶点 细胞 拓扑异构酶可以分为两个不同的组。 1型 酶通过在DNA螺旋的一条链中产生瞬时断裂而起作用， 使单链或双链DNA穿过断裂，并重新密封所述断裂。 休息 2型酶以类似的方式工作，除了它们产生 螺旋的两条链都有短暂的断裂。 这两种类型的例子 酶存在于真核生物和原核生物中。 DNA拓扑异构酶Ⅲ (Topo III）是已经从大肠杆菌分离的1型酶。 该酶的晶体结构已被确定， 活性拓扑异构酶结构。 这种拓扑异构酶是 它在拓扑异构酶中的独特之处在于它可以作用于DNA和RNA 印刷受体. 此外，已发现该酶的同系物是 由混杂的、广泛宿主范围的质粒基因组编码， 酵母和人类基因组。 到目前为止，E。大肠杆菌是唯一一个 生物化学和遗传学分析都适用的活性。 的 该补助金的具体目标是：1）评估第1类的拟议模式 拓扑异构酶介导的松弛和去连环化; 2）进一步鉴定 参与底物结合的Topo III多肽的关键残基 和DNA切割，并试图阐明 在分子水平上的酶机制; 3）试图识别细胞 与Topo III相互作用并绘制Topo III区域的蛋白质， 4）抑制topB表达并分析其对 其他拓扑异构酶基因的表达和 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.