CAREER: Establishment of Replication Timing and Patterning in Mammalian Cells

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Intellectual merit:The structure of chromatin, the complex of genomic DNA and proteins found in the nucleus of eukaryotic cells, governs cellular processes including DNA replication, transcription and post-transcriptional gene regulation. But, how that is accomplished is an important question that remains to be clearly elucidated. In mammalian cells, chromosomal domains occupy a specific nuclear position and replicate at defined times during S-phase of the cell division cycle. The spatiotemporal pattern of DNA replication within a given cell type is heritable and is established during the G1 phase of the cell cycle. There is, however, minimal understanding of the events and molecules that govern the spatiotemporal patterning of DNA replication. The Origin Recognition Complex (ORC) plays critical roles in the initiation of DNA replication, chromatin organization and cell cycle progression. The binding of the largest subunit of human ORC, Orc1 to chromatin is an obligatory step for pre-RC assembly during G1. Similarly, Orc2-5 display dynamic association with chromatin in a cell cycle-dependent manner. The interplay between ORC and chromatin structure that govern replication timing and patterning remain to be elucidated. The objective of the present project is to determine how ORC contributes to and/or reads epigenetic marks during G1. The project will address the role of ORC in establishing spatial patterning and timing of replication in G1 and determine the role of chromatin architecture in modulating ORC binding to chromatin. This research is innovative in its combination of molecular biology and biochemistry with state of the art live cell imaging. This project is highly significant as it aims to advance our understanding of how chromatin organization and replication are coordinated in living cells.Broader impacts:This research will have a strong and active outreach potential, since it utilizes extensive high-resolution microscopy as well as time-lapse live cell imaging that is crucial not only to understand key biological questions on replication, but also to serve as a platform for undergraduate students to appreciate biology. This approach will be applied in several ways including by teaching undergraduate students, by providing research opportunities to University of Illinois and community college students, by mentoring future researchers, and by organizing a symposium for students to appreciate the excitement of basic research. These activities will all involve women and minority students. This project integrates the use of Biochemistry, Cell Biology and Molecular Biology to capture the interest of students from varied backgrounds, including Biology, Physics, Chemistry and Bioengineering. With a small investment of time, exposing young and bright students to research early on in life will be highly beneficial and instill in them a sense of scientific awareness.