Investigation of DnaB helicase loading for initiating DNA replication

用于启动 DNA 复制的 DnaB 解旋酶负载研究

• 批准号: 8003692
• 负责人: Valerie Lynn O'Shea
• 金额: $ 5.05万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8003692
• 关键词: ATP Hydrolysis; Anti-Bacterial Agents; Binding; Biochemical; Biological Assay; Biological Models; Cell physiology; Complex; DNA; DNA Footprint; DNA biosynthesis; Data; Deposition; Detection; DnaB helicase; Event; Genetic; Investigation; Life; Methods; Molecular; Molecular Conformation; Organism; Plasmids; Play; Process; Proteins; Replication Initiation; Replication Origin; Resolution; Role; Single-Stranded DNA; Site-Directed Mutagenesis; Structure; System; Tertiary Protein Structure; Testing; base; chemotherapeutic agent; flexibility; helicase; melting; nucleotide protein interaction; public health relevance; recombinational repair

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.

描述（由申请人提供）：DNA解旋酶是多亚基、大分子机器，其使用来自ATP水解的能量来解链双链体片段以驱动基本细胞过程，如DNA复制、重组和修复。与真核生物和古细菌类似，细菌复制解旋酶DnaB在高度调控的DNA复制过程中起着许多重要作用，包括形成双向复制叉。为了帮助叉的形成，DnaB的六聚体环必须首先打开并沉积在熔化的复制起点的单链DNA区域上，这一过程取决于起始蛋白DnaA和DnaB装载配偶体DnaC。特异性蛋白质-蛋白质和蛋白质-核苷酸相互作用如何以方向特异性和链特异性方式促进两个DnaB六聚体的加载仍然是一个突出和重要的问题。使用生物化学和结构的方法，解旋酶加载的机制将通过确定复制起始子和解旋酶加载蛋白的精确作用以及它们在此过程中对ATP的使用来阐明。具体而言，将研究DnaA和DnaC依赖性与DnaB的相互作用在促进定向特异性解旋酶加载到熔化的复制起点的每个DNA链上的作用（目的1）。最近的数据表明，DnaA和DnaC在将两个DnaB六聚体装载到DNA上方面具有不同但互补的功能。为了测试这一想法，将使用基于质粒的生化DNA足迹分析，连同定点诱变，以探测DnaA，DnaB和DnaC之间的特异性相互作用如何差异地有助于两个解旋酶六聚体在两个互补链上的相反方向的加载。这种方法将允许检测解旋酶加载的DNA链特异性效应。此外，DnaB解旋酶与DnaA和DnaC相互作用的分子基础将使用结构方法定义（目的2）。DnaA和DnaC都是多结构域蛋白，其中关键功能模块通过柔性系链连接。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，并将这些片段用作入口以确定各自与DnaB的共晶结构。这些复合物的高分辨率结构将提供对支持解旋酶加载所需的相互作用的分子理解，以及DnaA或DnaC结合是否改变DnaB的构象。 公共卫生相关性：在生命的所有领域中，遗传稳定性取决于DNA复制起始的调节过程，这一事件需要适当加载两个解旋酶拷贝以形成双向复制叉。积累的数据表明，复制起始的一般过程是保守的所有生物体，包括需要复制启动子和解旋酶加载功能，然而，这些系统之间的相似性和差异尚未建立。该建议使用细菌复制作为模型系统，以阐明启动子和解旋酶加载蛋白在解旋酶加载中的互补但不同的作用，并将提供必要的分子细节，用于开发复制启动作为新的抗菌剂和化疗剂的目标。