The 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.