The elasticity of single DNA molecules and chromatin fibers determined by force-measuring laser tweezers.

摘要

Chromatin is the first step of DNA packing in eukaryotic cells. During the cell cycle the structure of chromatin undergoes dramatic changes in compaction in response to the needs of the cell. It has been known that the actively transcribing regions of the DNA are organized in looser, accessible structures, while the inactive regions remain packed in a compact structure. Understanding the elastic behavior of DNA and chromatin can provide insights about how these mechanical properties mediate these conformational changes and generate the first level of gene expression.; An optical tweezers force transducer was built to measure the elasticity of DNA and chromatin. Experimental results confirmed that the calibration for the light-momentum force transducer is independent of particle size, particle shape, focal depth, buffer index or laser power. A single DNA molecule or a single chromatin fiber was connected between two polystyrene beads, one held on a glass pipette and the other in the laser trap. The extension was measured by the position of the beads and the applied force was measured directly from the force transducer.; Under a longitudinal stress of ∼65 piconewtons (pN), dsDNA molecules in aqueous buffer undergo a reversible transition into a stable form with 5.8 Å rise per base pair, 70% longer than B-form dsDNA. This transition was affected by changes in ionic strength of the medium and water activity or by cross-linking of the two strands of dsDNA. Single-stranded DNA was also stretched giving a persistence length of 10 Å.; In 5 mM NaCl, the force-extension curves of chicken erythrocyte chromatin fibers display three distinct regions. Below 5 pN, the stretch and release curves are reversible. If the fiber is stretched beyond 10 pN, the extension cycle displays hysteresis but the process is repeatable. Tension higher than 20 pN damages the fiber. In 40–150 mM NaCl, a new, distinctive, transition in the force-extension curves appears below 6 pN which is likely due to internucleosomal attraction.; This dissertation includes both my previously published and my co-authored materials.