抄録
Eukaryotic DNA is packaged in nuclei into nucleosome, which is the basic unit of chromatin, wrapping around histone octamers. Like other nuclear activities involving replication and transcription, the regulation of DNA repair upon damage by irradiation are also regulated at the chromatin level. We have analyzed the kinetics of core histones in living cells using photobleaching techniques. This technique is now applied to analyze dynamics of histone variants during DNA damage and repair. To understand the molecular mechanism of histone exchange found in living cells, we developed an in vitro system that mimics in vivo events using permeablized cells so that the required components can be manipulated. HeLa cells were permeabilized with a non-ioninc detergent, and incubated in extracts prepared from cells expressing histones tagged with the green fluorescent protein (GFP). As is in living cells, H3-GFP and H4-GFP in extracts preferentially targeted to replicated chromatin in permeabilzed cells, and H2A-GFP and H2B-GFP to euchromatin regardless of DNA replication. Although purified H2A-H2B complex containing H2A-GFP failed to assemble into permeabilzed cell chromatin, its assembly was supported by the addition of cell extracts. Using this system, we expect that the factors essential for histone assembly and exchange can be identified and the physiological role of histone modification can be revealed. We hope this permeabilized cell system will be useful for understanding the regulation of DNA damage repair, as is photobleaching in living cells.
