Molecular Mechanisms of Bacterial Helicase Assembly and Activation at a Replication Origin

细菌解旋酶在复制起点组装和激活的分子机制本工作的目的是通过对细菌DNA复制起点解旋酶组装的基于结构的分析来了解DNA复制的一个基本特征。复制解旋酶将双链DNA解解成用于复制的模板。与对DNA沿单链DNA （ssDNA）易位的广泛理解相比，对解旋酶打开、在ssDNA上的解旋酶组装以及由解旋酶装载物介导的解旋酶关闭的机制知之甚少，本研究将解决这些问题。科学家-教师的主要任务是吸引和留住学生到STEM（科学、技术、工程和数学）领域，为21世纪培养受过良好教育的公民。纽约城市学院（CCNY）的另一个使命是为纽约市（NYC）的孩子，新移民，少数民族和那些没有经济能力的人提供高质量的教育。为了完成这些任务，PI将通过研究DNA复制（一个基本的生物过程）来指导和培训年轻的同事；2)领导本科生研究项目；3)对纽约市的四所公立学校进行教育推广，每一所学校都有相当多的少数族裔人口。分析细胞复制DNA的机制对于理解基因组是如何遗传和进化的至关重要。在大肠杆菌中，复制解旋酶是一个封闭的蛋白质环，其在单链DNA上的组装需要专门的装载因子。解旋酶的组装发生在DNA复制的起始阶段。这一阶段的主要事件包括选择开始复制的位点，融化起始DNA片段，然后将解旋酶装载到ssDNA上。解旋酶装载复合体首先捕获六聚体dna解旋酶，然后在原点与ssDNA接合。这导致装载者从原始复合体中排出，并伴随着螺旋酶的激活，采用闭合螺旋构象。解旋酶的平面和螺旋结构是已知的，但对解旋酶的加载机制知之甚少。研究人员已经确定了与噬菌体P解旋酶装载物结合的dna解旋酶的结构。该项目将侧重于从结构中获得的见解。从本研究中获得的结构见解，当与其他小组的见解相结合时，导致了细菌中解旋酶组装途径的新模型。下面的目的是试图扩展和测试从研究人员修订的模型中获得的见解。目标1侧重于评估和扩展从BP模型中获得的见解。在目标#2中，他们重点分析与起源衍生的ssDNA结合的BP复合体。他们将进一步探索其模型的一个特征：dna解旋酶虽然与解旋酶装载器结合，但可能很少与ssDNA发生接触。相反，大多数（如果不是全部的话）接触将由解旋酶加载器复合物内的加载器进行。在目标#3中，他们将注意力转向与BP复合体向噬菌体传递相关的机制&amp；#955；来源。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Molecular Mechanisms of Bacterial Helicase Assembly and Activation at a Replication OriginThe goal of this work is to understand an essential feature of DNA replication through a structure-based analysis of assembly of the bacterial DnaB replicative helicase at the origin of DNA replication. Replicative helicases unwind duplex DNA into templates for replication. In contrast to the extensive understanding of the translocation of DnaB along single stranded DNA (ssDNA), relatively little is known about the mechanisms of helicase opening, helicase assembly on ssDNA, and closing of the helicase as mediated by helicase loaders, which this research will address. A main mission of scientists-teachers is to attract and retain of students to STEM (science, technology, engineering and mathematics) fields and to prepare an educated citizenry for the 21st century. City College of New York (CCNY) has the additional mission of providing a high-quality education to the children of New York City (NYC), new immigrants, minorities, and those without economic means. To meet these missions, the PI will 1) mentor and train younger colleagues through research on DNA replication, a fundamental biological process; 2) lead undergraduate research programs; and 3) perform educational outreach to four public schools in NYC, each with a significant minority population.Analyses of mechanisms used by cells to replicate DNA are essential for understanding how genomes are inherited and evolve. In E. coli, the replicative helicase is a closed protein ring whose assembly on single stranded (ss) DNA requires specialized loading factors. Assembly of the helicase takes place during the initiation phase of DNA replication. The major events of this phase include selection of sites where replication will begin, melting of a segment of origin DNA and then loading the helicase on ssDNA. The helicase-loader complex first captures the hexameric DnaB helicase and then engages ssDNA at the origin. This leads to expulsion of the loader from the origin complex, and concomitant helicase activation with adoption of a closed spiral conformer. The planar and spiral structures of the helicase are known, however, less is known about the mechanisms of helicase loading. The investigators have determined the structure of the DnaB helicase bound to the P helicase loader from phage λ. This project will focus on insights obtained from the structure. Insights from the structure obtained in this research, when combined with those from other groups, lead to a new model of the helicase assembly pathway in bacteria. The aims below seek to extend and test insights obtained from the investigators' revised model. Aim #1 focuses on evaluating and extending insights obtained from the BP model. In aim #2, they pivot to analysis of the BP complex bound to origin derived ssDNA. They will further explore one feature of their model: that the DnaB helicase, while bound to the helicase loader, may make few, if any, contacts to ssDNA. Instead, most, if not all, of the contacts will be made by the loader within the helicase-loader complex. In aim #3, they turn their attention to mechanisms associated with delivery of the BP complex to the phage λ origin.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.