The research team will study the biophysical properties of specific chromosomal domains and the role forces play in their function throughout the cell cycle. DNA and RNA polymerases generate considerable force (up to 40pN) during processes of replication and transcription. During mitosis, microtubules attach to centromeric regions to provide the motive force for chromosome segregation. The tension generated by microtubule attachment (~20pN/microtubule) between sister centromeres of replicated chromosomes is critical to the mechanisms that ensure the fidelity of chromosome segregation upon anaphase onset. Thus mechanical force plays a critical role in DNA metabolic processes. However, excessive force (>10pN) can inhibit chromatin assembly in S-phase and breakage of chromosomes with two centromeres (dicentric chromosomes) in mitosis. It is therefore likely that forces on chromosomes are spatially as well as temporally regulated throughout the cell cycle. Recent experiments have addressed the amount of force required to stretch DNA and displace nucleosomes in vitro. These experiments reveal different DNA-protein interactions around the nucleosome core and enhance our understanding of the enzymatic processes that require access to nucleosomal DNA. In this project they will isolate specific chromatin domains and determine the biophysical properties of distinct regions of the chromosomes. They will apply force to chromatin to measure the force-extension relationships for centromeres, euchromatin and telomeric sequences. Intellectual Merit: Using this approach, they will dissect the DNA sequence and protein structural contributions to the biophysical properties of specific sub-chromosomal domains. In addition, they have identified proteins that recognize DNA under tension. By examining the force extension curves for specific chromatin domains in cells lacking these components they will establish the genetic requirements for specific force extension signatures. They expect this work to lead to a Force-extension map for an entire eukaryotic chromosome. This approach will provide the first biomechanical view of the chromosome and will be critical in understanding how energy and structural information is stored. Broader Impact: This research will be integrated into education and outreach through three venues: a networked molecular manipulation project to K12 students, through integration into an undergraduate science perspective course, and through an extensive undergraduate research program. The first program allows K12 students to manipulate real molecules (DNA, Viruses) under an AFM that is located remotely at UNC. In a typical year this program reaches over 200 K12 students, allowing undergraduates and graduate researchers the experience of mentoring and exciting K12 students. The science of forces in mitosis will be included as a section in a science perspective class, "How Things Work" taught to over 250 non-science majors each year. For undergraduate research, educational goals will focus on integrating research and teaching activities to give students the tools to evaluate and employ new technologies. About 6 students will study forces in mitosis within an intensive research-based program over the course of the grant.
