| Summary: | 碩士 === 國立臺灣大學 === 微生物學研究所 === 93 === We studied the roles of topoisomerase II (TOP2) isoforms in chromosomal DNA loop domain organization/function. The eukaryotic nuclear DNA is packed with histones to form nucleosome, and further partitioned into topological/ chromatin loops emanating from the nuclear matrix/scaffold. Interestingly, DNA TOP2 has been shown to be a structural component of mitotic chromosome scaffolds. Recently, two isoforms of TOP2 have been identified (TOP2α and TOP2β) in mammalian cells and in bacterial cells [gyrase and topoisomerase IV (Topo IV)]. Here, we used the RNA interference technology and other genetic approaches to create TOP2 isozyme-deficient conditions for studying the structural/enzymatic roles of TOP2 isoforms in organizing the chromosomal DNA loop domains. Both of the isozyme-specific, RNAi-knockdown clones had been isolated and verified by Western blot analysis. In addition, all of the RNAi knockdown clones confer resistance to TOP2-targeting drugs. Preliminary results using PFGE analysis have also suggested that TOP2β might play a more dominant role in loop organization.
To further support the notion that TOP2 is involved in the loop organization of the chromosome, we also explored the potential existence of chromosomal loops in prokaryotic cells. Our results showed that TOP2-targeting antibiotics can effectively cleave bacterial chromosome into 50-100 kb DNA fragments, indicating the existence of loop domains. Strains with genetic manipulations of drug-resistant gyrase and/or Topo IV provide a unique opportunity for dissecting their roles in bacterial chromosomal DNA loop organization. Using the loop-excision protocol and TOP2 drug-resistant bacteria, we have been able to identify gyrase, rather than TopoIV, as the main player in the organization of loop domains in bacterial nucleoid.
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