Investigation of DnaB helicase loading for initiating DNA replication

描述（由申请人提供）：DNA解旋酶是一种多亚基、大分子机器，它利用ATP水解产生的能量来解开双链片段，以驱动DNA复制、重组和修复等基本细胞过程。与真核生物和古细菌一样，细菌的复制解旋酶DnaB在高度调控的DNA复制过程中发挥着许多重要作用，包括形成双向复制叉。为了帮助叉的形成，必须首先打开DnaB的六聚体环，并将其沉积到熔化的复制起始点的单链DNA区域上，这一过程取决于启动蛋白DnaA和DnaB装载伙伴DnaC。特异性蛋白质-蛋白质和蛋白质-核苷酸相互作用如何以定向和链特异性的方式促进两种dna六聚体的装载仍然是一个突出而重要的问题。利用生物化学和结构方法，通过确定复制启动器和解旋酶装载蛋白在这一过程中的精确作用，以及它们对ATP的使用，来阐明解旋酶装载的机制。具体来说，我们将研究DNA和dnac依赖于DnaB的相互作用在促进定向特异性解旋酶装载到熔化复制起点的每条DNA链上的作用（目的1）。最近的数据表明，DnaA和DnaC在将两种DNA六聚体装载到DNA上具有不同但互补的功能。为了验证这一想法，将使用一种基于质粒的生化DNA足迹测定，以及位点定向诱变，来探测DNA、DnaB和DnaC之间的特定相互作用如何在不同的方向上促进两个解旋酶六聚体在融化源的两条互补链上的装载。这种方法将允许检测DNA链对解旋酶装载的特异性影响。此外，将使用结构方法定义DnaB解旋酶与DnaA和DnaC相互作用的分子基础（目标2）。DnaA和DnaC都是多结构域蛋白，其关键功能模块通过柔性链连接在一起。由于这种类型的分子构型会干扰结构研究，因此将建立足以与DnaB相互作用的DnaA和DnaC的最小区域，这些片段将用作确定它们与DnaB共晶结构的入口通道。这些共络合物的高分辨率结构将提供支持解旋酶装载所需的相互作用的分子理解，以及DnaA或DnaC结合是否改变了DnaB的构象。

DESCRIPTION (provided by applicant): DNA helicases are multi-subunit, macromolecular machines that use energy derived from ATP hydrolysis to unwind duplex segments to drive essential cellular processes such as DNA replication, recombination, and repair. Like its eukaryotic and archaeal counterparts, the bacterial replicative helicase, DnaB, plays many important roles in the highly-regulated process of DNA replication, including formation of a bidirectional replication fork. To assist in fork formation, the hexameric ring of DnaB must first be opened and deposited onto the single-stranded DNA regions of a melted replication origin, a process that depends on the initiator protein, DnaA, and the DnaB loading partner, DnaC. How specific protein-protein and protein-nucleotide interactions facilitate the loading of two DnaB hexamers in an orientation- and strand-specific manner remains an outstanding and important question. Using biochemical and structural methods, the mechanism of helicase loading will be elucidated by determining the precise roles of replication initiator and helicase loading proteins, and their use of ATP, in this process. Specifically, the role of DnaA- and DnaC-dependent interactions with DnaB in promoting the orientation-specific helicase loading onto each DNA strand of a melted replication origin will be investigated (Aim 1). Recent data suggest that DnaA and DnaC have distinct, but complementary, functions in loading two DnaB hexamers onto DNA. To test this idea, a plasmid-based, biochemical DNA footprinting assay will be used, together with site-directed mutagenesis, to probe how specific interactions between DnaA, DnaB, and DnaC differentially contribute to the loading of two helicase hexamers in opposing directions on the two complementary strands of a melted origin. This approach will allow for detection of DNA strand-specific effects on helicase loading. Additionally, the molecular basis for the interaction of DnaB helicase with DnaA and DnaC will be defined using a structural approach (Aim 2). DnaA and DnaC are both multi-domain proteins in which critical functional modules are connected by flexible tethers. As this type of molecular configuration can interfere with structural investigations, the minimal regions of both DnaA and DnaC sufficient to interact with DnaB will be established, and these fragments will be used as entryways to determine co-crystal structures of each with DnaB. High-resolution structures of these co-complexes will provide a molecular understanding of the interactions necessary to support helicase loading, and whether DnaA or DnaC binding alters the conformation of DnaB. PUBLIC HEALTH RELEVANCE: Among all domains of life, genetic stability is dependent upon the regulated process of DNA replication initiation, an event that requires proper loading of two copies of helicase for formation of a bidirectional replication fork. Accumulating data suggests that the general process of replication initiation is conserved among all organisms, including the need for replication initiator and helicase loader functionalities, however, the similarities and differences between these systems have not been established. This proposal uses bacterial replication as a model system to elucidate the complimentary yet distinct roles of initiator and helicase loader proteins in helicase loading, and will provide the molecular details necessary for developing replication initiation as a target for new antibacterial and chemotherapeutic agents.