CAREER: Dissecting the Mechanism of Replication Initiation in Vertebrates via Single Molecule Imaging

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This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2). This project will employ a cutting-edge microscopy approach to investigate the earliest stage in DNA replication at a molecular level. Several proteins act in concert to copy DNA bidirectionally from sites called “replication origins”. How this process initiates bidirectionally from thousands of origins remains one of the most mysterious aspects of DNA biology. This research will advance basic understanding of DNA replication mechanisms that safeguard the integrity of genetic information. The educational goals of this project are: (1) to collaborate with teachers from a local underserved high school to develop a hands-on biotechnology curriculum, (2) to provide professional training opportunities for high-school science teachers and research opportunities for high-school students, and (3) to develop skills training courses for graduate students. American Rescue Plan funding provides support for this researcher at a critical stage in his career.Replication initiation involves short-lived interactions between DNA and numerous proteins, which are challenging to monitor and measure using current approaches. Although the process of replication initiation has been studied in detail in yeast, key proteins are poorly conserved between yeast and vertebrates and their exact functions remain unclear. This project will systematically dissect replication initiation in the Xenopus egg extract model system using novel single-molecule imaging methodology developed by the principal investigator. This effort will be complemented by biochemical, genomic, and proteomic approaches. The research has the potential to advance the field by (1) providing a quantitative view of replication initiation in higher eukaryotes, (2) identifying and characterizing unknown regulators of replication initiation, and (3) elucidating why key replication proteins have dramatically increased in complexity from yeast to humans.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.