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

细菌解旋酶在复制起始处的组装和激活的分子机制本工作的目标是通过对细菌DNAB复制解旋酶在DNA复制起始处的组装进行基于结构的分析来了解DNA复制的基本特征。复制型解旋酶将双链DNA解离成模板进行复制。与DNAB沿单链DNA(SsDNA)转位的广泛了解相比，对于解旋酶的打开、解旋酶在单链DNA上的组装和解旋酶的关闭机制知之甚少，本研究将针对这些机制进行研究。科学家-教师的主要使命是吸引和留住学生进入STEM(科学、技术、工程和数学)领域，并为21世纪培养受过教育的公民。纽约城市学院(CCNY)的额外使命是为纽约市(NYC)的孩子、新移民、少数族裔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1)通过研究DNA复制(一个基本的生物学过程)来指导和培训年轻的同事；2)领导本科生研究计划；以及3)对纽约市的四所公立学校进行教育推广，每所学校都有相当多的少数人口。分析细胞复制DNA的机制对于了解基因组是如何遗传和进化至关重要的。在大肠杆菌中，复制解旋酶是一个闭合的蛋白质环，它在单链DNA上的组装需要特殊的负载因子。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复制开始的位置，融化一段起始DNA，然后将解旋酶加载到单链DNA上。解旋酶-加载器复合体首先捕获六聚体DNAB解旋酶，然后在起始处与单链DNA结合。这导致加载器从起始复合体中排出，并伴随着通过采用闭合螺旋构象而激活解旋酶。解旋酶的平面和螺旋结构是已知的，然而，对解旋酶的负载机制知之甚少。研究人员已经确定了与噬菌体P解旋酶载入器结合的DNAB解旋酶的结构。本项目将侧重于从该结构中获得的见解。从这项研究中获得的结构的见解，当与其他小组的见解相结合，导致了细菌中解旋酶组装途径的新模型。下面的目标试图扩展和检验从调查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，他们将重点放在分析与起源的单链DNA结合的BP复合体上。他们将进一步探索他们模型的一个特征：DNAB解旋酶虽然与解旋酶加载器结合，但可能很少与单链DNA接触。相反，大多数接触，如果不是全部，将由解旋酶-加载器复合体中的加载器进行。在目标3中，他们将注意力转向与将BP复合体交付给原生噬菌体相关的机制。这一裁决反映了NSF的法定使命，并通过使用基金会的智力优势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.