Single-Strand DNA-Binding Proteins and Deoxyribonucleotide Enzyme Organization

[603684] Mathews本项目测试DNA复制模型，其中DNA的脱氧核糖核苷三磷酸前体由与复制机制相连的酶复合物合成，从而允许将建筑材料有效地运送到DNA复制位点。在T4噬菌体感染的大肠杆菌中，dNTP生物合成酶形成一种复合物，称为dNTP合成酶，其中包含至少8种病毒编码酶和2种宿主细胞来源的酶。在体外，该配合物表现出动力学耦合，即能够有效地催化多步反应途径，而中间产物的积累很少。因此，该复合物在体外的表现就像它在体内必须表现的那样，如果它要充当DNA底物穿梭体，在复制位点附近合成dNTPs。从T4 dNTP合成酶复合体中纯化的重组酶已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。这提示了一种DNA复制模型，其中多个dNTP合成酶复合物被T4 SSB吸引到复制叉上。本项目将对该模型进行物理和酶动力学分析，分析复合物中蛋白质与T4基因32蛋白之间的关联；基因32蛋白在吸引dNTP合成酶到复制位点中的作用的研究；基因32蛋白酸性c端与dntp合成酶相互作用的研究；并试图重建部分dNTP合成酶复合物。如果资源允许，这项研究将扩展到牛痘病毒，它也编码一个与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有组织的蛋白质组装，这增加了细胞功能的效率，通过防止中间体扩散远离利用地点。由于DNA复制发生在有限数量的位点上（细菌中每条染色体只有两个复制叉），因此需要dNTP合成酶的组织，以足够快地将前体运送到这些少数位点，以支持观察到的高DNA复制率，并保持遗传信息传递的高精度。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。了解这种协调可能会导致对基本生物过程的细胞内组织的更好理解。

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.