• Journal of Microbiology and Biotechnology
• /
• 제25권10호
• /
• pp.1768-1771
• /
• 2015

Human Fen1 protein (hFen1) plays an important role in Okazaki fragment processing by cleaving the flap structure at the junction between single-stranded (ss) DNA and doublestranded (ds) DNA, an intermediate formed during Okazaki fragment processing, resulting in ligatable nicked dsDNA. It was reported that hChlR1, a member of the cohesion establishment factor family, stimulates hFen1 nuclease activity regardless of its ATPase activity. In this study, we found that cohesion establishment factors cooperatively stimulate endonuclease activity of hFen1 in in vivo mimic condition, including replication protein-A-coated DNA and high salt. Our findings are helpful to explain how a DNA replication machinery larger than the cohesion complex goes through the cohesin ring structure on DNA during S phase in the cell cycle.

• 한국자기공명학회논문지
• /
• 제22권3호
• /
• pp.71-75
• /
• 2018

The CRISPR-Cas system is the adaptive immune system in bacteria and archaea against invading phages or foreign plasmids. In the type II CRISPR-Cas system, an endonuclease Cas9 cleaves DNA targets of phages as directed by guide RNA comprising crRNA and tracrRNA. To avoid targeting and destruction by Cas9, phages employ anti-CRISPR (Acr) proteins that act against host bacterial immunity by inactivating the CRISPR-Cas system. Here we report the backbone $^1H$, $^{15}N$, and $^{13}C$ resonance assignments of AcrIIA4 that inhibits endonuclease activity of type II-A Listeria monocytogenes Cas9 and also Streptococcus pyogenesis Cas9 using triple resonance nuclear magnetic resonance spectroscopy. The secondary structures of AcrIIA4 predicted by the backbone chemical shifts show an ${\alpha}{\beta}{\beta}{\beta}{\alpha}{\alpha}$ fold, which is used to determine the solution structure.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1995년도 춘계학술대회
• /
• pp.80-80
• /
• 1995

T4 Endonuclease V (Mw 16,000) acts as a repair enzyme for UV induced pyrimidine dimers in DNA. Many researchers have studied the biochemical characteristics of the enzyme. However the precise action mechanism of T4 endo V has not fully elucidated yet. In our laboratory NMR spectroscopy technique is being used for the structural study of T4 endo V. Because of its low temperature stability and high content of ${\alpha}$-helix, the conventional $^1$H NMR technique was inapplicable. Therefore we utilized stable isotope labeling technique and so far prepared about 10 amino acid specific labeled proteins. The HSQC spectra of amino acid specific labeled proteins will help us to interpret the triple resonance 3D, 4D data which are under processing, We also studied the behaviors of specific amino acid residues whose roles might be critical. When the enzyme labeled by $\^$15/N-Thr was mixed with the substrate oligonucleotide (semispecific -TT- sequence), one crosspeak in its HSQC spectrum was completely desappeared, which means that one of seven Thr residues is in the binding site of the enzyme with DNA, This result is well consistent with previous report that implicated the Thr 2 residue in the activity of the enzyme. Similar studies were carried on the behaviors of Arg and Tyr residues.

• Bulletin of the Korean Chemical Society
• /
• 제35권10호
• /
• pp.3005-3008
• /
• 2014

Human ChlR1 protein (hChlR1), a member of the cohesion establishment factor family, plays an important role in the segregation of sister chromatids for maintenance of genome integrity. We previously reported that hChlR1 interacts with hFen1 and stimulates its nuclease activity on the flap-structured DNA substrate covered with RPA. To elucidate the relationship between hChlR1 and Okazaki fragment processing, the effect of hChlR1 on in vitro nuclease activities of hFen1 and hDna2 was examined. Independent of ATPase activity, hChlR1 stimulated endonuclease activity of hFen1 but not that of hDna2. Our findings suggest that the acceleration of Okazaki fragment processing near cohesions may aid in reducing the size of the replication machinery, thereby facilitating its entry through the cohesin ring.

• The Plant Pathology Journal
• /
• 제17권1호
• /
• pp.57-61
• /
• 2001

Phytoplasmas were identified from two chrysanthemum (Dendranthema grandiflorum) plants showing different symptoms ; one with stusting, rosette, and excessive branching (Ph-ch1), and the other with stunting and chlorosis (Ph-ch2). Electron microscopy of midrib of the plants with the symptoms revealed that numerous phytoplasmas were localized in the phloem cells. The disease was transmitted from infected plants to healthy ones by grafting. Phytoplasma-specific DNA was detected in polymerase chain reaction (PCR) analysis with template DNA extracted from the leaves of Ph-ch1 and Ph-ch2, both of which yielded a same DNA band corresponding to 1.5 kb. Using a specific primer pair (R16F1/R1) synthesized based on aster yellows (AY) phytoplasma, a DNA fragment of 1.1 kb was amplified by PCR. Endonuclease restriction patterns of the 1.1 kb PCR products from Ph-ch1 and Ph-ch2, which were dgeste with each of the restriction endonucleases Sau3A, Hha, Alu and Rsa, were same as those of AY phytoplasma from periwinkle. This suggests that the chrysanthemum plants (Ph-ch1 and Ph-ch2) be infected with a phytoplasma belonging to AY phytoplasma.

• 한국어병학회지
• /
• 제8권1호
• /
• pp.31-36
• /
• 1995

HcNPV DNA genome 을 제한효소 EcoRI 으로 절단하여 그들의 일부 절편을 pUC8 vector 에 cloning 한 후 E. coli JM 83 세포에 형질 전환시켰다. 이 결과 24 개의 EcoRI 절편중 12 개의 절편이 cloning 되었다. 이들 제조합체중 4 개는 eNP-O, eNP-Q, eNP-R, eNP-S 라 명명하였다. 또한 이들 제조합체의 외래 유전자 발현을 SDS-PAGE 에 의해 단백질 패턴을 분석하였다. 그 결과 제조합체 eNP-O, eNP-Q, eNP-R 에서는 E. coli JM 83 숙주세포의 단백질 밴드와 비교하여 다른 분자량을 갖는 밴드가 나타났다.

• International Journal of Stem Cells
• /
• 제16권2호
• /
• pp.234-243
• /
• 2023

The recent advances in human pluripotent stem cells (hPSCs) enable to precisely edit the desired bases in hPSCs to be used for the establishment of isogenic disease models and autologous ex vivo cell therapy. The knock-in approach based on the homologous directed repair with Cas9 endonuclease, causing DNA double-strand breaks (DSBs), produces not only insertion and deletion (indel) mutations but also deleterious large deletions. On the contrary, due to the lack of Cas9 endonuclease activity, base editors (BEs) such as adenine base editor (ABE) and cytosine base editor (CBE) allow precise base substitution by conjugated deaminase activity, free from DSB formation. Despite the limitation of BEs in transition substitution, precise base editing by BEs with no massive off-targets is suggested to be a prospective alternative in hPSCs for clinical applications. Considering the unique cellular characteristics of hPSCs, a few points should be considered. Herein, we describe an updated and optimized protocol for base editing in hPSCs. We also describe an improved methodology for CBE-based C to T substitutions, which are generally lower than A to G substitutions in hPSCs.

• 미생물학회지
• /
• 제32권3호
• /
• pp.208-214
• /
• 1994

토양으로부터 분리한 방선균주로부터 새로운 type II 제한효소를 분리하여 그 특성을 연구하였다. 이 균주는 수리분석 결과, Streptoverticillium olivoverticillatum으로 동정되었으며, 정제한 제한효소는 SolI이라 명명하였다. SoII은 BamHI의 isoschizomer로서 여섯 개의 염기서열 5'-G $\downarrow$ GATCC- 3'을 인지하며 두 개의 G 염기 사이에서 절단하여 4 base가 돌출된 5'말단을 생성한다. 그러나 BamHI과는 달리, dam methylation 되어 있는 인식 염기서열에는 작용하지 못하였다. Ammonium sulfate 분획(30-65%)과 heparin-agarose, Affi-gel Blue column chromatography를 거쳐 SolI을 부분 정제하였다. SolI은 활성을 보이기 위하여 0.2mM 이상의 $MgCl_2$를 반드시 필요로 하였으며, 다른 cofactor는 요구하지 않았다. NaCl이 없을 때 가장 높은 활성을 가지며 120 mM 이상의 NaCl이 있으면 활성이 완전히 억제되었다. 이 효소의 반응 최적 온도는 $40^{\circ}C$, 최적 pH는 8.6으로 나타났다. Gel filtration chromatography에서의 용출부피 비교로부터 이 효소의 분자량은 약 43,000Da인 것으로 추정된다.

• 미생물학회지
• /
• 제32권1호
• /
• pp.91-95
• /
• 1994

토양에서 분리된 Enterobacter agglomerans CBNU45는 type II 제한효소인 EagBI을 생산하고 있음을 발견하였다. EagBI을 DEAE-cellulose, phosphocellulose P11, hydroxylapatite column chromatography를 거쳐 부분 정제하여 그 특성을 알아보았다. EagBI은 여섯개의 염기배열 5’-CGAT${\downarrow}$CG-3’을 인식하고 T 와 C 사이를 절단하여 두개의 염기가 3’-말단쪽으로 돌출된 cohesive end를 형성하였다. EagBI의 반응 최적조건은 10mM Tris-HCl(pH 7.8), 6~10mM $MgCl_2$, 37${\circ}C$이었으며 NaCl이 없는 반응 완충용액에서 가장 좋은 활성을 보였다. EagBI은 $dam^-$와 $dam^+$ 메칠화 DNA도 절단할 수 있으며 65${\circ}C$에서 10분 동안 열처리하였을 때 효소의 활성을 상실하였다. 따라서 EagBI은 PvuI의 isoschizomer이나 NaCl 요구성과 열안정성에서 PvuI보다 편리한 제한효소로 확인되었다.

• Journal of electromagnetic engineering and science
• /
• 제15권3호
• /
• pp.142-150
• /
• 2015

Several reports supported that continuous exposure to 60 Hz magnetic field (MF) induces testicular germ cell apoptosis in vivo. We recently evaluated duration- and dose-dependent effects of continuous exposure to a 60 Hz MF on the testes in mice. BALB/c male mice were exposed to a 60 Hz MF at $100{\mu}T$ for 24 hours a day for 2, 4, 6, or 8 weeks, and at 2, 20 or $200{\mu}T$ for 24 hours a day for 8 weeks. To induce the apoptosis of testicular germ cell in mice, the minimum dose is $20{\mu}T$ at continuous exposure to a 60 Hz MF for 8 weeks, and the minimum duration is 6 weeks at continuous exposure of $100{\mu}T$. Continuous exposure to a 60 Hz MF might affect duration- and dose-dependent biological processes including apoptotic cell death and spermatogenesis in the male reproductive system of mice. The safety guideline of the International Commission on Non-Ionizing Radiation Protection (ICNIRP) indicates that the permissible maximum magnetic flux density for general public exposure is $200{\mu}T$ at 60 Hz EMF (ICNIRP Guidelines, 2010). In the present study, we aimed to examine the expression of pro- and anti-apoptotic genes regulated by the continuous exposure to 60 Hz at $200{\mu}T$ in Sprague-Dawley rats for 20 weeks. The continuous exposure to 60 Hz at $200{\mu}T$ does not affect the body and testicular weight in rats. However, exposure to 60 Hz MF significantly affects testicular germ cell apoptosis and sperm count. Further, the apoptosis-related gene was scrutinized after exposure to 60 Hz at $200{\mu}T$ for 20 weeks. We found that the message level of endonuclease G (EndoG) was greatly increased following the exposure to 60 Hz at $200{\mu}T$ compared with sham control. These data suggested that 60 Hz magnetic field induced testicular germ cell apoptosis through mitochondrial protein Endo G.