科研之友
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摘要
All genomic transactions in eukaryotes take place in the context of the nucleosome, the basic unit of chromatin, which is responsible for DNA compaction. Overcoming the steric hindrance that nucleosomes present for DNA-processing ezymes requires significant conformational changes. The dynamics of these have been hard to resolve. Single-pair Fluorescence Resonance Energy Transfer (spFRET) microscopy is a powerful technique for observing conformational dynamics of the nucleosome. Nucleosome immobilization allows the extension of observation times to a limit set only by photobleaching, and thus opens the possibility of studying processes occuring on timescales ranging from milliseconds to minutes. It is crucial however, that immobilization itself does not intoduce artifacts in the dynamics. Here we report on various nucleosome immobilization stratergies such as single-point attachment to polyethylene glycol (PEG) or surfaces coated with bovine serum albumin (BSA), and confinement in porous agarose or polyacrylamide gels. We compare the immobilization specificity and structural integrity of immobilized nucleosomes. A cross-linked star polyethylene glycol coating perrforms best with respect to tethering specificity and nucleosome integrity, and enables us to reproduce for the first time bulk nucleosome unwrapping kinetics in single nucleosomes without immobilization artifacts.
- 出版日期2008-10-6
