Investigation of DnaB helicase loading for initiating DNA replication

描述(申请人提供)：DNA解旋酶是一种多亚基的大分子机器，它使用来自ATP水解的能量来解开双链片段，以驱动DNA复制、重组和修复等基本的细胞过程。与真核生物和古生物一样，细菌复制解旋酶DNAB在DNA复制的高度调控过程中发挥着许多重要作用，包括形成双向复制叉子。为了帮助分叉的形成，DNAB的六聚体环必须首先被打开并沉积在熔化的复制起点的单链DNA区域上，这一过程取决于启动子蛋白DNAA和DNAB的装载伙伴DNAC。特定的蛋白质-蛋白质和蛋白质-核苷酸相互作用如何促进两个DNAB六聚体以定向和链特异的方式负载仍然是一个悬而未决的重要问题。利用生化和结构方法，通过确定复制启动子和解旋酶负载蛋白在这一过程中的确切作用以及它们对ATP的使用，将阐明解旋酶负载的机制。具体地说，将研究依赖于DNAA和DNAC的与DNAB的相互作用在促进定向特定的解旋酶加载到熔融复制起点的每条DNA链上的作用(目标1)。最近的数据表明，Dna A和Dna C在将两个Dna B六聚体装载到DNA上的过程中具有不同但互补的功能。为了验证这一想法，我们将使用基于质粒的生化DNA足迹试验，结合定点突变，来探索Dna A、Dna B和Dna C之间的特定相互作用如何差异地导致两个解旋酶六聚体在相反方向上负载在融化来源的两条互补链上。这种方法将允许检测DNA链特异性对解旋酶负载的影响。此外，将使用结构方法确定DNAB解旋酶与DNAA和DNAC相互作用的分子基础(目标2)。DNAA和DNAC都是多结构域蛋白质，其中关键的功能模块通过灵活的系链连接。由于这种类型的分子构型可能会干扰结构研究，因此将建立足以与DNAB相互作用的DNAA和DNAC的最小区域，并且这些片段将被用作确定每个片段与DNAB的共晶结构的入口。这些共复合物的高分辨结构将使人们从分子上了解支持解旋酶负载所必需的相互作用，以及DNAA或DNAC结合是否改变了DNAB的构象。 与公共健康相关：在生命的所有领域中，遗传稳定性取决于DNA复制启动的调控过程，这一事件需要适当加载两个副本的解旋酶才能形成双向复制叉子。越来越多的数据表明，复制起始的一般过程在所有生物体中都是保守的，包括复制启动子和解旋酶加载器功能的需要，然而，这些系统之间的相似和不同之处尚未确定。这项建议使用细菌复制作为一个模型系统来阐明启动子和解旋酶负载器蛋白在解旋酶负载中互补但不同的作用，并将提供必要的分子细节来开发复制起始作为新的抗菌和化疗药物的靶点。

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.