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3DFM Magnetic Force System Detects Individual Nucleosome Disruption in Chromatin

DNA contains the information to code for all of the proteins in the human body. While itself a long chain molecule, DNA is found in cells in highly compacted forms. This compaction is a highly regulated system, and is exposed to forces during cell division and transcription that would unfold DNA and expose it to enzymes. The role of these forces in gene expression and regulation may be important in genetic defects. DNA Chromatin, the condensed form of DNA, is made up of DNA and histone proteins. The association of DNA with these histones forms the nucleosome, a structure that condenses the DNA by wrapping it 1.65 times around a histone octomer. The histone octomer is made up of two copies of each of histones H2A, H2B, H3 and H4, and is known as the Nucleosome Core Particle (NCP). Further compaction of the DNA is accomplished through interactions between core histone N-terminal domains and linker histones. This structure, and conformational changes that may take place throughout the cell cycle, is important to gene regulation and understanding the mechanisms behind transcription, replication and repair.

Chromatin fibers were manipulated and fiber extension was monitored, with specific attention paid to sudden increases in overall extension, an indication of a possible nucleosome disruption event. Three consecutive extensions of the same fiber are shown above. For the initial application of force, where the maximum applied force was approximately 15 pN, the tension on the nucleosome (histone-DNA complex) was not large enough to cause a disruption event. The overall extension of the chromatin fiber was significantly less (< 50%) than what would be expected for b-form DNA alone, indicating that the nucleosome organization of the fiber remained intact. In the second extension of the fiber, with a maximum force of approximately 24 pN, one nucleosome disruption event was observed during the ‘hold’ interval. Using this method, the amplitude of this disruption event was determined to be 51 nm. For the third extension of the fiber (max force approximately 30 pN), three nucleosome disruption events were observed, with amplitudes of 144 nm, 68 nm, and 68 nm for the 1st 2nd and 3rd events. This data was the basis of the successful grant application of Prof. Kerry Bloom to NSF, funded in 9/2005.