| Summary: | 碩士 === 國立中興大學 === 生命科學院碩士在職專班 === 92 === TypeⅡA DNA topoisomerase are involved in all aspects of DNA metabolism including replication, transcription, recombination, and chromosome segregation. This group of enzymes manipulates DNA topology by generating a transient double-stranded break on a DNA duplex(referred as gate-forming or simply G-segment), passing the other duplex(termed transport or T-segment)through the break, and re-ligating the G-segment. The quaternary structure of all known typeⅡA topoisomerases exhibit two-fold symmetry, with two ATPase domains forming the DNA entry gate, two Toprim/CAP-like folds mediating the ATP-dependent cleavage of G-segment, and a primary dimerization region also functioning as exit gate for T-segment. In bacteriophage T2, topoisomerase Ⅱ is composed of two gene products:T2-39 for ATPase and Toprim domains, and T2-52 for CAP-like fold and the DNA exit gate. Although the structural feature of the exit gate appears to be conserved for all type ⅡA enzymes, the mechanistic roles of this observation has not yet been addressed; important molecular events such as ATP binding and hydrolysis, DNA trapping, cleavage and religation all occur in other spatially distant regions. To answer this question, we constructed an exit gate deletion mutant T2-52 using SOE PCR. The mutant protein was successfully expressed and purified to apparent homogeneity. Two assays were performed as the index for typeⅡtopoisomerase activity:supercoiled DNA relaxation and kinetoplast DNA decatenation. Surprisingly, the deletion mutant was active in both assays. We also used typical type Ⅱ topoisomerase inhibitors, vanadate and m-AMSA to analyze the mutant’s sensitivity spectrum. Although the mutant was more sensitive to the inhibitor, the patterns were essentially the same. The implication of the exit gate in typeⅡ topoisomerase catalysis will be addressed in my thesis.
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