Single-Strand DNA-Binding Proteins and Deoxyribonucleotide Enzyme Organization

9603684马修斯这个项目测试了一种DNA复制模型，在该模型中，DNA的脱氧核糖核苷三磷酸前体是由连接到复制机器的酶复合体合成的，从而使建筑材料能够有效地运送到DNA复制位置。在T4噬菌体感染的大肠杆菌中，dNTP生物合成的酶形成一个复合体，称为dNTP合成酶，它包含至少八种病毒编码的酶和两种宿主细胞来源的酶。在体外，该络合物表现出动力学偶联，即能够有效地催化多步反应途径，并且几乎不积累中间体。因此，该复合体在体外的行为必须像它在体内的行为一样，如果它要作为DNA底物穿梭，在复制位置附近合成dNTP。从T4dNTP合成酶复合体中纯化的重组酶被证明直接或间接地与基因32编码的T4单链DNA结合蛋白(SSB)结合。这提出了一种DNA复制模型，在该模型中，多个dNTP合成酶复合体通过对T4 SSB的吸引力而被吸引到复制叉上。本项目将通过对复合体中的蛋白质与T4基因32蛋白之间的结合进行物理和酶动力学分析；研究基因32蛋白在将dNTP合成酶吸引到复制位置中的作用；研究基因32蛋白的酸性C末端对与dNTP合成酶的相互作用的作用；以及尝试重建部分dNTP合成酶复合体来测试该模型。如果资源允许，这项研究将扩展到痘苗病毒，它还编码一种与DNA前体合成酶相互作用的SSB。许多细胞代谢过程被发现涉及到有组织的蛋白质组装，这通过防止中间产物从利用部位扩散而提高了细胞功能的效率。由于DNA复制发生在有限的位置(细菌中每个染色体只有两个复制叉)，因此特别需要组织dNTP合成的酶，足够快地将前体递送到这些少数位置，以支持观察到的高DNA复制率，并在遗传信息传输中保持高精度。此外，由于dNTPs除了用作DNA前体外，没有已知的代谢作用，因此这些前体的合成必须与DNA复制密切协调。理解协调可能会导致更好地理解基本生物过程的细胞内组织。

9603684 Mathews This project tests a model of DNA replication in which the deoxyribonucleoside triphosphate precursors to DNA are synthesized by enzyme complexes linked to the replication machinery, allowing thereby the efficient delivery of building materials to DNA replication sites. In T4 bacteriophage-infected Escherichia coli, the enzymes of dNTP biosynthesis form a complex, called dNTP synthetase, which contains at least eight virus-coded enzymes and two enzymes of host cell origin. In vitro, the complex displays kinetic coupling, i.e., the ability to catalyze multi-step reaction pathways efficiently and with little accumulation of intermediates. Thus, the complex behaves in vitro as it must behave in vivo if it is to serve as a DNA substrate shuttle, synthesizing dNTPs in the immediate vicinity of replication sites. Purified recombinant enzymes from the T4 dNTP synthetase complex have been shown to associate, directly or indirectly, with the T4 single-strand DNA-binding protein(SSB) encoded by gene 32. This suggests a model for DNA replication in which multiple dNTP synthetase complexes are drawn to replication forks by their attraction for T4 SSB. This project will test that model by physical and enzyme kinetic analysis of association among proteins in the complex and the T4 gene 32 protein; investigations of the role of gene 32 protein in drawing dNTP synthesizing enzymes to replication sites; investigation of the role the acidic C-terminus of gene 32 protein upon interactions with dNTP-synthesizing enzymes; and attempted reconstitution of parts of the dNTP synthetase complex. If resources permit, this investigation will be extended to vaccinia virus, which also encodes an SSB that interacts with enzymes of DNA precursor synthesis. Many cellular metabolic processes are being found to involve organized protein assemblies, which increase the efficiency of cellular function by preventing diffusion of intermediates away from sites of utilization. Because DNA replication occurs at a limited number of sites (as few as two replication forks per chromosome in bacteria), there is a special need for organization of enzymes of dNTP synthesis, to deliver precursors to those few sites rapidly enough to support observed high rates of DNA replication and to maintain high accuracy in the transfer of genetic information. Moreover, because dNTPs have no known metabolic roles other than their use as DNA precursors, the synthesis of those precursors must be closely coordinated with DNA replication. Understanding that coordination may lead to a greater understanding of the intracellular organization of a fundamental biological process.