SSB PROTEINS AS ORGANIZERS OF DNA PRECURSOR BIOSYNTHESIS

描述：该提案将研究DNA之间的协调 复制及其前体脱氧核糖核苷的合成 三磷酸（dNTPs）。 在T4噬菌体感染的E.大肠杆菌、dNTP酶 生物合成形成称为dNTP合成酶的复合物，其至少含有 八种病毒编码的酶和两种宿主细胞酶， 体外偶联 dNTP合成酶与 复合物和多蛋白质DNA复制复合物尚未定义， 而这种关系正是这一提议的主旨所在。 复合物中的八种酶已被纯化为重组体 蛋白质并固定在色谱柱上。 T4蛋白分析 结合显示每个柱保留gp 32，单链DNA结合 由T4基因编码的蛋白质（SSB）32。 一个简单的想法是，gp 32的行为， 作为连接器，将dNTP合成酶复合物吸引到复制叉， 可能在每个分叉的dNTP合成酶的多个拷贝中。 由于gp 32结合 单链模板DNA链刚好在子DNA之前 3 '-羟基末端，这种缔合将dNTP合成酶精确地定位在 需要使DNA复制发生的空间饱和。 类似的情况可能存在于被牛痘病毒感染的细胞中， 单链特异性DNA结合蛋白，与病毒编码的 已经描述了核糖核苷酸还原酶。 用固定化蛋白质和DNA-纤维素进行亲和层析， 非变性凝胶电泳和免疫沉淀将用于 区分dNTP合成酶复合物中的那些蛋白质， 与gp 32直接相互作用。 的影响 纯化的gp 32在dNTP合成酶之间的物理和动力学缔合 酶将被探测。 表面等离子体共振将被分析， 定量结合亲和力、化学计量和协同性。 扰动 基因32在体内的功能将被测试其对关联的影响。 dNTP合成酶与DNA-蛋白质复合物的分离感染 细胞 为了测试模型的普遍适用性，牛痘病毒SSB将 进一步分析。 条件表达式系统的开发将 允许实验来定义蛋白质的代谢作用， 支持DNA复制并可能绘制病毒编码的dNTP 合成酶复制位点。 亲和层析， 固定化的p34及其C-末端截短衍生物，将允许 鉴定其蛋白质关联。 表征 SSB的磷酸化将探索这种修饰对 蛋白质-蛋白质和蛋白质-DNA关联。

DESCRIPTION: This proposal will study the coordination between DNA replication and the synthesis of its precursors, the deoxyribonucleoside triphosphates (dNTPs). In T4 phage-infected E. coli, the enzymes of dNTP biosynthesis form a complex, called dNTP synthetase, which contains at least eight viral-encoded and two host-cell enzymes and which shows kinetic coupling in vitro. The physical relationship between the dNTP synthetase complex and the multi-protein DNA replication complex has not been defined, and that relationship represents a main thrust of this proposal. Eight of the enzymes in the complex have been purified as recombinant proteins and immobilized on chromatographic column. Analysis of T4 proteins bound shows that each column retains gp32, the single-stranded DNA-binding protein (SSB) encoded by T4 gene 32. A simplifying idea is that gp32 acts as a connector, drawing the dNTP synthetase complex to the replication fork, possibly in multiple copies of dNTP synthetase per fork. Since gp32 binds single-stranded template DNA strands just ahead of the daughter DNA 3'-hydroxyl terminus, this association would place dNTP synthetase precisely where it is needed to saturate the space where DNA replication is occurring. A similar situation may exist in cells infected with vaccinia virus, where a single-strand-specific DNA-binding protein that interacts with viral-encoded ribonucleotide reductase has been described. Affinity chromatography with immobilized proteins and DNA-cellulose, non-denaturing gel electrophoresis, and immunoprecipitation will be used to distinguish those proteins in the dNTP synthetase complex that interact directly with gp32 from those that interact indirectly. The effects of purified gp32 upon physical and kinetic association among dNTP synthetase enzymes will be probed. Surface plasma resonance will be analyzed to quantitate binding affinities, stoichiometry, and cooperativity. Perturbing gene 32 function in vivo will be tested for its effects on the association of dNTP synthetic enzymes with DNA-protein complexes isolated from infected cells. To test the general applicability of the model, the vaccinia virus SSB will be further analyzed. Development of a conditional expression system will permit experiments to define the metabolic roles of the protein, in supporting DNA replication and possibly in drawing viral-encoded dNTP synthetic enzymes to replication sites. Affinity chromatography, of immobilized p34 and its C-terminal truncated derivatives, will permit identification of its protein associations. Characterization of the phosphorylation of SSB will explore the role of this modification on protein-protein and protein-DNA associations.