DETERMINATION OF SEQUENCE SPECIFICITY BETWEEN A PLASMID REPLICATION INITIATOR PROTEIN AND THE ORIGIN OF REPLICATION
DETERMINATION OF SEQUENCE SPECIFICITY BETWEEN A PLASMID REPLICATION INITIATOR PROTEIN AND THE ORIGIN OF REPLICATION
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DOI：
10.1006/jmbi.1995.0625
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发表时间：
1995-12-01
影响因子：
5.6
通讯作者：
SHAW, WV
中科院分区：
文献类型：
--
作者：
THOMAS, CD;NIKIFOROV, TT;SHAW, WV
pT 181家族的葡萄球菌质粒通过滚环机制复制，需要质粒特异性Rep蛋白的活性。起始事件涉及复制起点ori内Rep的位点特异性磷酸二酯键裂解。在体外的Rep蛋白也显示I型拓扑异构酶活性特异性的这个质粒家庭虽然键裂解，ICR II，是保守的pT 181家族的所有成员之间的单一位点，质粒特异性Rep蛋白能够区分家庭成员在体内，启动复制只从同源的起源。这种特异性的基础被认为是由于一个非共价键之间的相互作用的Rep和DNA序列相邻的磷酸二酯键cleaving.Using质粒pC 221指定的RepD蛋白质，我们目前的RepD：oriD复合物形成的物理参数的数据。通过凝胶迁移率变动实验测量的不同但相关的ori靶序列的非共价相互作用的相对强度的定量已经产生了与体内蛋白质的已知特异性雅阁的数据。寡核苷酸竞争实验表明，这种相互作用确实是由于特异性决定簇，ICR III。蛋白质-DNA交联方法表明RepD的羧基末端蛋白水解片段与其同源复制起点的ICR III区域发生特异性相互作用。拓扑异构酶率的分析表明，ICR III和蛋白质的羧基末端之间的相互作用是必需的生产性的相互作用，即在ICR II的磷酸二酯键裂解之前，可以发生。(C)1995年学术出版社
Staphylococcal plasmids of the pT181 family replicate by a rolling circle mechanism, requiring the activities of a plasmid-specified Rep protein. The initiation event involves site-specific phosphodiester bond cleavage by Rep within the replication origin, ori. In vitro the Rep proteins also display type-I topoisomerase activity specific for this plasmid family Although the single site of bond cleavage, ICR II, is conserved among all members of the pT181 family, the plasmid-specific Rep proteins are able to discriminate between family members in vivo, initiating replication only from the cognate origin. The basis of such specificity is believed to be due to a non-covalent binding interaction between Rep and a DNA sequence adjacent to the site of phosphodiester bond cleavage.Using the RepD protein specified by plasmid pC221, we present data for the physical parameters of RepD:oriD complex formation. Quantification of the relative strengths of the non-covalent interactions for different but related ori target sequences, measured by gel mobility shift experiments, has yielded data that are in accord with the known specificity of the protein in vivo. Oligonucleotide competition experiments demonstrate that this interaction is indeed attributable to the specificity determinant, ICR III. Protein-DNA crosslinking methods show that a carboxyl-terminal proteolytic fragment of RepD makes a specific interaction with the ICR III region of its cognate replication origin. Analysis of topoisomerase rates indicates that the interaction between ICR III and the carboxyl terminus of the protein is required before a productive interaction, namely the phosphodiester bond cleavage at the ICR II, can occur. (C) 1995 Academic Press Limited