DNA Supercoiling: Local and Global Aspects

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DESCRIPTION (provided by applicant): Understanding chromosome organization and its control of gene expression represents one of the most fundamental open biological challenges. Genomic organization and expression are intimately related because the complex structure and dynamics of DNA and protein-bound DNA at a large range of spatial and temporal scales regulate basic processes of life, such as the movement of mobile genetic elements ("mobile DNA") like plasmids or transposons, and transcription initiation. Mobile DNAs are transferred among bacterial pathogens and can propagate bacterial pathogenicity (through virulence genes), as well as drug resistance. While the acquisition of mobile DNAs is only the first of many stages in the evolution of specialized pathogens such as plague, E. coli, cholera, and anthrax, it has been hypothesized that common mechanisms are responsible for regulating intercellular gene transfer in many pathogens. In eukaryotic transcription, the first step in protein synthesis, RNA synthesis can only proceed when the DNA is accessible: through a complex network of nucleosome modifications, variant histones, and remodeling, it is hypothesized that eukaryotic genomes alter states of folding and compaction of the chromatin fiber to control DNA access and, as such, orchestrate (recruit or repress) transcription as needed. Many details of mobile DNA transfer and chromatin organization are unknown. The goal of the proposed work is to elucidate structural/dynamical mechanisms associated with such regulatory control in the transfer of mobile DNAs within genomes and in chromatin organization following histone modifications. Our long-term goals are to integrate structural and dynamics aspects of chromatin organization and regulation with transcription initiation to delineate thermodynamic mechanisms involved. Both processes combine regulatory local protein/DNA interactions with global responses in large systems of protein-bound supercoiled DNA. Based on models and applications completed under prior support, we will integrate protein/DNA conformations at atomic resolution on the nanosecond scale with global aspects of site juxtaposition in supercoiled DNA on the millisecond scale through mesoscale models (which incorporate local details where needed and macroscopic features where possible). Our aims are to: 1) test/delineate the local conformational changes hypothesized to repress transposition in specific transposase monomer, dimer, and inhibitor complexes; 2) determine effects of plasmid superhelicity, DNA interwound conformations, and mobile DNA size on site synapsis times and juxtaposition mechanisms in very long DNA, and thereby estimate the probability of DNA transfer to complement traditional biochemical and genetic approaches to propagation of microbial virulence; and 3) delineate hypothesized crucial electrostatic effects of two sequence variants of histone H2 and modified tails of histones H3 and H4 on chrornatin organization. Resulting insights can ultimately be exploited to design conditions that might limit mobile DNA propagation and hence microbial pathogen spread, or affect transcription initiation, such as enzymes that regulate DNA super coiling and nucleosome composition and pharmaceutical compounds that interfere with synaptic complexes and chromatin folding/unfolding rearrangements.