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The repeat structure, nucleosome, is composed of ~147 base pairs genome DNA wrapped by a histone octamer (H2A, H2B, H3 and H4) in ~1.7 left-handed helical turns, and the universal structure is responsible for the DNA organization and compaction in eukarya. However, chromosomal DNA should be dynamically regulated to ensure the proper regulation of gene transcription, replication, repair and recombination inside cells. It has been reported that there are at least five different ways to regulate the chromosomal structure in vivo: the variants replacement (eg: H2A.Z), post-translation modification of histone (PTMs), DNA methylation, the ATP-independent (eg: histone chaperones) and ATP-dependent (nucleosome remodelers) chromatin remodeling. Therefore, we would like to combine novel optical nanotools, spectroscopy assay and biochemistry assay to investigate the chromosomal organization in vitro in order to obtain the detailed molecular mechanism.
Histone assembly process
Histone assembly process
A DNA molecule without specific sequence or with DNA601 sequence (high affinity for histone) is anchored on the coverglass, and the other end of DNA molecules is tethered with a streptavidin-labeled nanoparticle. The change in the movements of tethered bead can reflect the change in the DNA length. The histone chaperone-mediated nucleosome assembly process can be studied by analyzing the movements of tethered beads . Two different histone chaperones were purified, and their functions were investigated using TPM method.
Nucleosome remodeling process
Nucleosome remodeling process
In the beginning, TPM is used to study the yRSC-mediated octamer transfer. A nanoparticle is tethered by a 800 bp mononucleosome duplex DNA with 147 bp DNA601 sequence (High affinity for histone). The whole process will be investigated under different reaction coditions( w/o ATP, w/o accepter DNA, w/o histone chaperone).
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