자리 지시된 돌연변이 유발연구에 의한 제한효소 EcoRV의 금속 특이성 변화

다국어 초록 (Multilingual Abstract)

Asp 90 of the EcoRV protein has been substituted by Cys by site - directed mutagenesis to understand the metal ion specificity of the EcoRV enzyme.
At λ DNA cleavage assay, the D90C mutant showed about 100 times less activity than wild - type EcoRV at standard reaction condition and so it identified that Asp 90 was essential for the catalytic fimction of EcoRV. According to expectation, the D90C mutant showed higher activity at pH 8.8, which ionized the SH group of Cys, than at pH 7.5. In the presence of Mg^++, the wt EcoRV cleaved DNA with higher activity than the D90C mutant did, but in the presence of Mn^++, the mutant had higher activity than the wild - type EcoRV enzyme had.
One of the aims of this work was to attempt to make a mutant which has high Zn metal activity. However, the D90C mutant in the presence Zn^++ at pH 8.8 cleaved the recognition site of pAT plasmid DNA with activities that were close to that of the wild - type, but it showed much lower activities, compared to the presence of Mg^++ or Mn^++. The relative activities of D90C mutant enzyme on metal ions were Mn^++＞ Mg^++ ＞＞ Zn^++. Interestingly, the D90C mutant EcoRV showed higher sequence specificity than wt EcoRV, so it did not cleave nonspecific site of DNA even under star condition which has an organic solvent, DMSO.
The D90C mutant EcoRV showed that the mechanism of DNA cleavage by the mutant was differed from that of the wild type enzyme. In contrast to the wild type EcoRV, which cuts both strands of the DNA at its recognition site simultaneously, the mutant cuts DNA by the two step cleavage reactions. The mutant cut initially just one strand of double strands of DNA at its recognition site and then made linear DNA by the cleavage of the remaining strand in two sequential reactions.

목차 (Table of Contents)

• 목차
• I. 서론 = 1
• 1.1 EcoRV restriction enzyme의 구조와 기능 = 1
• 1.2 EcoRV restriction enzyme의 cleavage mechanism = 7
• 1.3 EcoRV overproduction과 protein purification = 10
• 1.4 연구 목적 = 10
• II. 실험 재료 및 방법 = 12
• 2.1 실험 재료 = 12
• 2.2 Single-stranded template DNA의 분리 = 13
• 2.3 Mutagenesis = 16
• 2.3.1 Phosphorylation of mutant oligonucleotide = 16
• 2.3.2 Mutagenesis = 16
• 2.4 Transformation = 19
• 2.5 Plasmid miniprep = 20
• 2.6 Positive clones의 감별 = 21
• 2.7 Mutant EcoRV gene의 활성화와 발현 = 21
• 2.7.1 Stuffer fragment의 제거 = 21
• 2.7.2 Mutant EcoRV gene의 발현 = 23
• 2.8 Mutant EcoRV 제한효소의 정제 = 23
• 2.9 Phosphocellulose P11 culumn의 준비 = 26
• 2.10 λ DNA cleavage assay = 27
• 2.11 pAT 153 cleavage assay = 27
• III. 실험 결과 및 고찰 = 29
• 3.1 Mutant oligonucleotide의 설계 = 29
• 3.2 Single-stranded template DNA 분리 = 30
• 3.3 Cla I Digesion = 30
• 3.4 Mutant EcoRV의 정제 = 30
• 3.5 λ DNA cleavage assay = 33
• 3.6 pAT 153 cleavage assay = 36
• 3.6.1 Mutant D90C EcoRV의 cleavage mechanism = 36
• 3.6.2 Metal ion에 따른 activity의 비교 = 40
• 3.7 Noncognate site에서의 반응 = 40
• 3.8 pAG DNA cleavage assay = 44
• IV. 결론 = 49
• V. 참고 문헌 = 51
• VI. 영문 초록 = 56