SSB PROTEINS AS ORGANIZERS OF DNA PRECURSOR BIOSYNTHESIS

描述：这项提议将研究DNA之间的协调 其前体脱氧核糖核苷的复制和合成 三磷酸盐(Dntp)。在T4噬菌体感染的大肠杆菌中，dNTP的酶 生物合成形成一种复合体，称为dNTP合成酶，它至少包含 八种病毒编码的酶和两种宿主细胞酶，这表明 体外偶联。DNTP合成酶之间的物理关系 复合体和多蛋白DNA复制复合体尚未定义， 而这种关系代表了这项提议的一个主旨。 该复合体中的8种酶已纯化为重组酶 蛋白质，固定在色谱柱上。T4蛋白的分析 Bound显示每一列都保留了gp32，即单链DNA结合 T4基因32编码的蛋白(SSB)。一个简化的想法是gp32起作用。 作为连接器，将dNTP合成酶复合体拉到复制叉上， 可能在每个分叉的dNTP合成酶的多个副本中。由于gp32绑定了 单链模板DNA链恰好在女儿DNA之前 3‘-羟基末端，这种结合将精确地放置dNTP合成酶 需要它来饱和发生DNA复制的空间。 在感染牛痘病毒的细胞中也可能存在类似的情况，其中 与病毒编码基因相互作用的单链特异性DNA结合蛋白 核糖核苷酸还原酶已被描述。 固定化蛋白质和DNA-纤维素的亲和层析， 非变性凝胶电泳和免疫沉淀将用于 区分dNTP合成酶复合体中相互作用的蛋白质 直接与来自那些间接交互的那些gp32。的影响 纯化的gp32对dNTP合成酶物理和动力学结合的影响 将会对酶进行探测。表面等离子体共振将被分析为 量化结合亲和力、化学计量比和协作性。心烦意乱 32基因在体内的功能将被测试其对关联的影响 病原菌DNA-蛋白质复合体与dNTP合成酶的比较 细胞。 为了测试模型的普遍适用性，痘苗病毒SSB将 有待进一步分析。条件表达系统的开发将 允许进行实验来定义蛋白质的代谢作用，在 支持DNA复制，并可能绘制病毒编码的dNTP 将合成酶转移到复制位置。亲和层析 固定化的p34及其C端截短的衍生物将允许 鉴定其蛋白质组合。刻画人物形象 SSB的磷酸化将探索这种修饰在 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.