The repeating structural unit of eukaryotic chromatin, the nucleosome, is composed of two copies each of the histone proteins H2A, H2B, H3, and H4. These proteins form an octamer around which 147bp of DNA is wrapped in 1.65 superhelical turns (Luger et al., 1997). The nucleosome represents a major obstacle for any protein seeking access to the DNA. Several strategies have evolved to regulate access to nucleosomal DNA, such as posttranslational modification of histones and histone variants, ATP-dependent chromatin remodeling machines, and histone chaperones. It is likely that most if not all of these mechanisms directly impact the thermodynamics of the nucleosome. The DNA sequence itself may also impact its own inherent accessibility through modulating nucleosome positioning and/or thermodynamics. However, these working hypotheses could not be tested directly because no assays to measure nucleosome stability under physiological conditions were available. Attempts to determine the stability of nucleosomes have been hampered by the fact that the nucleosomes do not assemble in vitro under physiological conditions, but will only form nucleosomes through titration from high (2M) to low (>0.3M) ionic strength. We have developed a coupled equilibrium approach using the histone chaperone Nap1 to measure nucleosome thermodynamics under physiological conditions. This method will be useful for examining the impact of DNA sequence, histone variants, and posttranslational modifications on nucleosome thermodynamics.
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