Structure/Function Studies of DNA Replication Initiation

描述（由申请人提供）：遗传物质的忠实繁殖对生命至关重要。DNA复制的一个核心先决条件是在染色体上的适当位置专门组装复制机器。这种复制“起始”事件取决于多种成分，包括：1)启动物，结合起始点并招募其他蛋白质到复制起始点的atp酶；2)解旋酶装载物，将复制解旋酶沉积到DNA上；3)引物酶，产生短寡核苷酸以供DNA聚合酶延伸的酶。我们研究的长期目标是了解控制DNA复制起始的分子结构/功能关系。许多负责起始的蛋白质已经被确定，它们的作用的初步框架已经到位。然而，关于这些因素如何单独和合作地构建一个合格的复制分叉，仍然存在许多悬而未决的问题。我们之前在这一领域的努力已经产生了引发剂作用、解旋酶加载和启动的新机制模型，我们现在处于一个理想的测试位置。利用结构、生化和生物物理方法的结合，我们的目标是：1)确定ATP如何通过原核启动物控制DNA结合和重塑，2)确定ATP如何调节细菌解旋酶装载物的组装和功能，以及3)确定细菌引物酶如何合成引物并被不同类型的小分子试剂（包括严格的反应调节剂ppGpp）抑制。总之，这些努力将影响许多科学前沿，从了解起源如何参与和重组以促进复制体的构建，到定义化学能和小分子抑制剂如何控制动态大分子机器的功能。了解这些过程对于理解细胞如何最终避免与基因组不稳定、转化和肿瘤相关的起始错误是必要的。公共卫生相关性：遗传物质的忠实繁殖对生命至关重要。我们的研究目标是在分子水平上理解控制DNA复制起始的结构/功能关系。了解这些事件对于理解细胞如何最终避免与基因组不稳定性和癌症发病相关的错误是必要的。

DESCRIPTION (provided by applicant): The faithful propagation of genetic material is essential to life. A central prerequisite to the copying of DNA is the dedicated assembly of replicative machineries at proper sites on the chromosome. This replication "initiation" event depends on multiple components, including: 1) initiators, ATPases that bind origins and recruit other proteins to the replication start site, 2) helicase-loaders, which deposit replicative helicases onto DNA, and 3) primases, enzymes that create short oligonucleotides for extension by DNA polymerases. A long-term objective of our research has been to understand the molecular structure/function relationships that govern the initiation of DNA replication. Many of the proteins responsible for initiation have been identified, and a preliminary framework for their action is in place. However, there remains a host of outstanding questions regarding how these factors act individually and cooperatively to construct a competent replication fork. Our prior efforts in this area have generated new mechanistic models for initiator action, helicase loading, and priming that we are now in an ideal position to test. Using a combination of structural, biochemical, and biophysical methods we aim to: 1) Define how ATP controls DNA binding and remodeling by prokaryotic initiators, 2) Determine how ATP regulates bacterial helicase-loader assembly and function, and 3) Establish how the bacterial primase synthesizes primers and is inhibited by disparate types of small-molecule agents, including the stringent response regulator, (p)ppGpp. Together, these efforts will impact a number of scientific fronts, from understanding how origins are engaged and restructured to promote replisome construction, to defining how chemical energy and small-molecule inhibitors control the function of dynamic macromolecular machines. Knowledge of these processes is necessary for understanding how cells ultimately avoid initiation errors linked to genomic instabilities, transformation, and neoplasia. PUBLIC HEALTH RELEVANCE: The faithful propagation of genetic material is essential to life. The goal of our research is understand the structure/function relationships that govern the initiation of DNA replication at a molecular level. Knowledge of these events is necessary for understanding how cells ultimately avoid errors linked to genomic instabilities and cancer onset.