| Popis: |
All living cells must organize their DNA into dynamic three-dimensional architectures that are compatible with essential cellular processes such as transcription, translation, and DNA repair. While this organization fundamentally affects such broad phenomena as antibiotic resistance, cell division, and the aggressiveness of cancer cells, DNA organization remains poorly understood due to the complexity of the many protein-DNA interactions involved. Recently it has been shown that DNA is able to condense into a crystalline lattice in bacterial cells exposed to stressful conditions. The single protein responsible for creating these DNA ‘biocrystals’ is Dps (DNA-binding protein from starved cells). When present at sufficiently high concentrations, Dps drives the condensation of DNA into a biocrystal both in vitro and in vivo. Here we investigate the energetics and kinetics that determine biocrystal formation. We have developed a novel single molecule assay to probe the physical interactions between fluorescently tagged Dps and DNA. Long DNA molecules were immobilized on a surface to study their spatial distribution during Dps-DNA biocrystal formation using total internal reflection fluorescence microscopy (TIRF) and epifluorescence microscopy. In addition, due to strong inter-particle interactions between Dps, the protein can form a two-dimensional crystal even in the absence of DNA. This two-dimensional crystal may represent an intermediate on the pathway to biocrystal formation. We have measured key features of this structure from atomic force microscopy (AFM) studies of Dps and DNA. From these data, we present a model for biocrystal formation. |
