Experimental and theoretical analysis of principles underlying molecular and genomic mechanobiology

About This Grant

Project Summary/Abstract Mechanistic understanding of living things requires our understanding of how proteins and DNA interact together to generate functional chromosomes. The structure and dynamics of chromosomes ultimately controls all functions of cells, and in turn, multicellular organisms, including humans. Understanding chromosome structure and dynamics and the underlying biochemical interactions defining them are central to preserving human health, dealing with genetic disorders, and fighting pathogenic organisms. Dramatic reorganizations of chromosomes occur throughout the cell cycle: in humans, hundred-million-base-pair long DNAs are genetically deactivated and refolded into the metaphase form to facilitate mitosis, following which are reorganized into cell nuclei harboring once again active gene expression. My laboratory studies chromosome structure and dynamics using a novel combination of cell- and molecule-scale mechanics with state-of-the-art genetic, biochemical, single-molecule and mathematical modeling tools. Chromosome mechanics at the nanonewton scale are central to cell division due to large mitotic spindle forces, and the well-defined elasticity of chromosomes also provides a quantitative readout of internal structural changes. Those micron-scale dynamic reorganizations of chromosomes are controlled by piconewton forces and nanometer steps generated by individual protein machines. Direct mechanistic analysis of chromosome organizational principles and their relation to underlying molecular interactions will transform our understanding of how cells interpret, fold and change their genomes. In turn this will advance understanding of pathologies where those functions are impaired including genetic disorders and cancers and will improve our understanding of how to target those functions in pathogenic organisms. Over the next five years my laboratory will analyze roles Structure of Maintenance of Chromosomes protein complexes (SMCs: condensin, cohesin and SMC5/6 in eukaryotes) and other key genome-acting proteins in organizing chromosomes across the three kingdoms of life, using single- molecule mechanics approaches to directly observe their function. In parallel we will use chromosome and nuclear mechanics studies to study their roles in organizing chromatin at the larger scales of metaphase chromosomes and cell nuclei. The remarkable stability of DNA-protein complexes will be studied using single- molecule and cell-level experiments on “facilitated dissociation” (FD), preliminary studies for which indicate that pathways for spontaneous dissociation – the backbone of our understanding of biochemical interactions – may be kinetically irrelevant compared to competitive binding pathways. This promises a complete revision of how we think about binding affinity in the crowded, competing in vivo environment, replacing the concept of a ligand-receptor affinity with a large competition kinetic matrix, with transformative implications for how we think about regulation of biochemical interaction networks in vivo. Experimental results will be linked to mathematical models and coarse-grained computer simulations of molecular function and genome/chromosome folding.