초록 ▼

- 극한 미생물들로부터 내열성 DNA polymerase 11 종 유전자에 대해 분자생물학적 • 생화학적 연구들을 완료.
- Neq DNA polymerase가 고온에서만 protein trans- splicing이 일어남을 실험적으로 증 명하였고, Neq L 단편과 Neq S 단편을 전구체 형태로 하나로 융합시켜 hot start PCR에 최적화된 Neq HS DNA polymerase의 개발함.
- 내열성 Tpa DNA polymerase와 Twa DNA polymerase를 이용하여 wild-type polymera

- 극한 미생물들로부터 내열성 DNA polymerase 11 종 유전자에 대해 분자생물학적 • 생화학적 연구들을 완료.
- Neq DNA polymerase가 고온에서만 protein trans- splicing이 일어남을 실험적으로 증 명하였고, Neq L 단편과 Neq S 단편을 전구체 형태로 하나로 융합시켜 hot start PCR에 최적화된 Neq HS DNA polymerase의 개발함.
- 내열성 Tpa DNA polymerase와 Twa DNA polymerase를 이용하여 wild-type polymerase보다 PCR 속도, DNA 증폭능력, proof reading 활성이 향상된 돌연변이체(Tpa-S N213D/K501R, Twa N501R)를 제조하여 PCR에의 응용.
- 초고온성 고세균 Crenarchaeota로부터 DNA ligase 유전자 3종의 특성을 조사한 결과 Szi 와 H bu DNA ligase의 경우, ATP외에도 ADP, GTP를 이용하는 복수의 cofactor specificity를 보임. 이러한 발견을 기초로 하여 생화학적 • 계통분류학적 연구들을 완료하여 cofactor specificity를 이용한 DNA ligase의 특성분석 및 진화적 연관관계를 규명.
- 신규 저온성 uracil-DNA glycosylase 유전자 3종에 대해 분자생물학적 • 생화학적 특성을 조사한 후 carry-over contamination 방지를 위한 PCR 응용에 관한 연구를 수행.
- 신규 내열성 Sma PPase와 Tpa dUTPase을 유전자들을 확보하여 이들 유전자에 대해 유전자 발현, 발현된 효소의 정제 • 특성조사 등 분자생물학적 • 생화학적 연구들을 완료

Abstract ▼

The purpose of this study is to develop useful DNA modifying enzymes, for using researches and diagnoses, from various marine and extreme microorganisms. ① The eleven genes coding for DNA polymerase were cloned and sequenced from hyperthermophilic extreme microorganisms (Sulfophobococcus zilligii (S

The purpose of this study is to develop useful DNA modifying enzymes, for using researches and diagnoses, from various marine and extreme microorganisms. ① The eleven genes coding for DNA polymerase were cloned and sequenced from hyperthermophilic extreme microorganisms (Sulfophobococcus zilligii (Szi), P yrobaculum arsenaticum (P ar), T. guaymasensis (Tgu), T. peptonophilus (Tpe), T. marinus (Tma), T. celer (Tce), T. pacificus (Tpa), T. radiotolerans (Tra), T. celericrescens (Tcel), T. waiotapuensis (Twa), T. coalescens (Tco)). The genes encoding thermostable DNA polymerases were expressed under the control of the T7lac promoter in E. coli. The purified DNA polymerases were biochemically characterized, and its application in PCR successfully demonstrated, respectively. ② Moreover, the two Nanoarchaeum equitans (Neq) DNA polymerase genes (Neq L and Neq S) were cloned and expressed individually, together, and as a genetically protein splicing-processed form. The expressed Neq DNA polymerases were purified and biochemical characterized. The small fragment (Neq S) of Neq DNA polymerase has no catalytic activity and the large fragment (Neq L) has lower 3´→5´ exonuclease activity; whereas the full-length Neq DNA polymerases have polymerase and 3´→5´ exonuclease activities, indicating that both fragments are needed to form the active DNA polymerase that possesses higher proofreading activity. It was demonstrated that natural protein trans-splicing occurs between the two fragments of Neq DNA polymerase at high temperature. Neq DNA polymerase can successfully catalyze PCR in the presence of dUTP.
These results suggest that Neq DNA polymerase could be most effectively utilized in real-time PCR using uracil-DNA glycosylase without the risk of carry-over contamination.
③ The site-directed mutant (Tpa-S N213D/K501R and Twa N501R) were constructed from Tpa DNA polymerase and Twa DNA polymerase to improve the industrial usefulness of DNA polymerase. ④ Finally, by fusing the Neq S fragment to Neq L fragment, Neq HS DNA polymerase was obtained. To improve PCR efficiency, mutant Neq HS DNA polymerases were created via site-directed mutagenesis. Nucleotide substitution from the Neq HS DNA polymerase was performed at A523R (Neq HS M1 DNA polymerase), A523R/N540R (Neq HS M2 DNA polymerase), and A523R/N540R/S185D (Neq HS M3 DNA polymerase). The protein splicing of Neq HS DNA polymerase and mutant Neq HS DNA polymerases were mainly dependent on the reaction temperature and occurred effectively at 70-95℃. Specifically, the triple mutant Neq HS M3 DNA polymerase (A523R/N540R/S185D) was more efficiently amplified various human genomic targent DNA than other mutant Neq HS DNA polymerases, Taq, P fu, and Taq HS DNA polymerases.
⑤ The genes encoding three hyperthermophilic DNA ligases (Staphylothermus marinus (Sma), Sulfophobococcus zilligii (Szi), H yperthermus butylicus (H bu)) were expressed under the control of the T7lac promoter in E. coli. The purified DNA ligases were biochemically characterized. Szi and H bu DNA ligases exhibited multiple cofactor specificity utilizing ADP and GTP in addition to ATP. The exploitation of GTP as a catalytic energy source has not to date been reported in any known DNA ligase. This phenomenon may provide evolutionary evidence of the nucleotide cofactor utilization by DNA ligases. We contend that DNA ligase evolution likely started from crenarchaeotal DNA ligases and diverged to eukaryal DNA ligases and euryarchaeotal DNA ligases. Subsequently, the NAD+-utilizing property of some euryarchaeotal DNA ligases may have successfully differentiated to bacterial NAD+-dependent DNA ligases. ⑥ The genes encoding four psychrophilic uracil-DNA glycosylases (UDGs) (P hotobacterium aplysiae GMD509 (P ap), P sychrobacter submarinus (P su), P sychrobacter sp. HJ147 (P sp), Bacillus sp. HJ171 (Bsp)) were expressed under the control of the T7lac promoter in E. coli. The purified uracil-DNA glycosylases were biochemically characterized. These cold-active characteristics enabled psychrophilic uracil-DNA glycosylases to control carryover contamination in PCR without losing the PCR product. ⑦ The genes encoding DNA modifying enzymes (Sma PPase, Tpa dUTPase) were expressed under the control of the T7lac promoter in E. coli. The purified DNA ligases were biochemically characterized.