Structural and Functional Analysis of the Initiation of DNA Replication in Bacteria

这项研究的长期目标是详细了解大分子复合物的结构和作用机制，这些大分子复合物在细菌起源的DNA复制中起作用。细胞通过在称为DNA复制起点的特殊位置组装大的蛋白质复合物来启动染色体DNA的复制。起源处的复合体通过因子的招募和解离进行重塑，最终成熟为一对完全形成的复制叉。在细菌中，第一个与起源结合的蛋白质是dna蛋白质，其中五个单体在不同的位置相互作用，形成一个大的核蛋白复合物。人们对这个建筑群的结构知之甚少。在ATP的作用下，该复合体促进DNA双链起始区域的解绕，从而使DnaB复制解旋酶（在DnaC蛋白的协助下）得以加载。在细菌、古细菌和真核生物中，在DNA复制起源处操作的组装体之间的相似之处，可以通过共同的机制细节和组成蛋白质中氨基酸序列的相似性来表明。这些相似之处有力地表明，对细菌提供的不那么复杂的复制系统的研究将产生共同的结构原理。PI将进行生化、生物物理和结构研究，以了解DNA蛋白在细菌DNA复制起始中的作用。尽管目前的理解水平有所提高，但关于DNA复制起源时DNA组装的结构和作用机制，仍有几个有趣的问题没有得到解答。这项研究将被整合到哈佛大学分子和细胞生物学系PI正在进行的科学和教育活动中。其中包括研究生和本科生的教育以及实验生物化学和结构生物学领域博士后的培养。

The long-term goal of this research is a detailed understanding of the structure and mechanism of action of the macromolecular complexes, which operate at the bacterial origin of DNA replication. Cells initiate replication of chromosomal DNA by assembling large protein complexes at specialized sites termed origins of DNA replication. The complexes at the origin undergo remodeling through the recruitment and dissociation of factors and eventually mature into a pair of fully formed replication forks. In bacteria, the first to protein to bind to the origin is the DnaA protein, five monomers of which interact at distinct sites and form a large nucleo-protein complex. The architecture of this complex is poorly understood. With ATP, this complex promotes the unwinding of regions of the DNA duplex at the origin, which enables loading of the DnaB replicative helicase (assisted by the DnaC protein). Parallels between the assemblies that operate at the origins of DNA replication in bacteria, archaea and eukaryotes are indicated by common mechanistic details and by amino acid sequence similarities in the constituent proteins. These parallels strongly suggest that common structural principles will emerge from studies with the less complicated replication systems offered by bacteria. The PI will perform biochemical, biophysical and structural studies to understand the role of the DnaA protein in the initiation of DNA replication in bacteria. Although the current state of understanding is advanced, several intriguing questions remain unanswered about the architecture and mechanism of action of the DnaA assembly at the origin of DNA replication. This research will be integrated into the scientific and educational activities being performed by the PI within the Department of Molecular and Cellular Biology at Harvard University. These include the education of graduate and undergraduate students and the training of post-doctoral fellows in the fields of experimental biochemistry and structural biology.