Graphene Oxide-based Direct Measurement of DNase I Activity with Single Stranded DNA

강종백 대한화학회 2014 Bulletin of the Korean Chemical Society Vol.35 No.9

Recent studies have shown that single-stranded DNA adsorbed onto graphene oxide is protected from DNase I cleavage. However, double-stranded DNA bound to graphene oxide and could be digested by DNase I. To elucidate whether single-stranded DNA is protect from DNase I in the presence of graphene oxide, this study conducted DNase I digestion using single-stranded DNA and single-stranded DNA containing the duplex region in the presence of graphene oxide. Addition of DNase I resulted in restoration of the fluorescence emission that had been quenched when DNA was adsorbed to graphene oxide. It indicates that DNase I cleaved the adsorbed single-stranded DNA onto graphene oxide, which was sufficient for the detection of DNase I activity.

Mutant cAMP Receptor Protein Binds to DNA without DNA Bending

강종백,Gang, Jong-Back Korean Society of Life Science 2006 생명과학회지 Vol.16 No.7

cAMP와 복합체를 형성한 cAMP 수용성 단백질은 DNA와 결합하여 ${\sim}90$도 정도의 예리한 DNA bending을 유도한다. 그러나 이전의 논문[5]에 의하면 돌연변이 CRP:cGMP 복합체는 돌연변이 CRP:cAMP 복합체보다 아크릴아미드 겔에서 상대적으로 빠른 이동속도를 보였다. CRP와 cyclic nucleotide 존재하에서 DNA의 구조 변화를 알아보기 위하여 6가지 준비된 DNA조각들을 사용하여 몰 고리화 인자(molar cyclization factor)[13]를 측정하였다. 이들 자료를 사용하여 nonlinear regression analysis를 통하여 cGMP 존재하에서 돌연변이 CRP는 DNA bending을 형성하지 않으나 CAMP 존재하에서 나선 꼬임과 같은 DNA 구조 변화없이 DNA bending을 형성한다. Cyclic AMP receptor protein (CRP) complexed with cAMP binds to DNA and induces sharp DNA bending around ${\sim}90$ degree. Previous publication (5), however, reported that mutant CRP:cGMP complex showed high migration rate relative to mutant CRP:cAMP complex on native polyacrylamide gel. To confirm DNA structural change in the presence of CRP and cyclic nucleotide, molar cyclization factor $(j_M)$ [13] was measured with 6 constructed DNA fragments. Nonlinear regression analysis of $j_M$ data indicated that mutant CRP did not induce DNA bending in the presence of cGMP but bent DNA in the presence of cAMP without any helical twist change in DNA.

Simple and Sensitive Fluorescence Assay of Restriction Endonuclease on Graphene Oxide

강종백 대한화학회 2015 Bulletin of the Korean Chemical Society Vol.36 No.9

Restriction endonucleases hydrolyze internal phosphodiester bonds at specific sites in a DNA sequence. These enzymes are essential in a variety of fields, such as biotechnology and clinical diagnostics. It is of great importance and necessity for the scientific and biomedical use of enzymes to measure endonuclease activity. In this study, graphene oxide (GO) has been used as a platform to measure enzyme activity with high sensitivity. To increase the detection sensitivity of Hinf I, the endonuclease-digested reaction was treated with exonuclease III (Exo III) and a fluorescence assay was conducted to measure the emission. Results showed that Exo III treatment enhanced 2.7-fold signal-to-background ratio for the detection of Hinf I compared with that done without Exo III in the presence of GO.

Proteus mirabilis 전사 조절 단백질의 DNA 결합 특성

강종백,Gang, Jong-Back 한국미생물학회 2011 미생물학회지 Vol.47 No.2

Proteus mirabilis 전사 조절($\underline{P}$roteus $\underline{m}$irabilis $\underline{t}$ranscription $\underline{r}$egulator ) 단백질의 중금속 결합 부위에 대한 아미노산 서열분석에서 PMTR 단백질은 ZntR (아연 저항성) 단백질이 아닌 CueR (구리 저항성) 단백질과 동일한 환경이다. 그리고 겔시프트 법(gel shift assay) 실험에 의하면 PMTR 단백질은 Escherichia coli의 zntA (zinc-translocating P-type ATPase gene) 프로모터에 결합하지 않고 copA (copper-translocating P-type ATPase gene) 프로모터와 Proteus mirabilis에서 atpase (copper-translocating P-type ATPase gene) 프로모터에 결합하였다. DNase I protection 실험에서 PMTR 단백질 결합부위와 DNase I 민감성 염기들이 관찰되었다. P. mirabilis atpase 프로모터에서 민감성 염기로 주형가닥(template strand)에서 C와 A 그리고 비주형가닥(non-template strand)에서 G와 C 염기들이다. 이런 민감성 염기들은 다른 MerR 패밀리 단백질에서 또한 관찰되었으며, 이것은 단백질에 의한 DNA bending을 의미한다. Amino acid sequence alignment shows that $\underline{P}$roteus $\underline{m}$irabilis $\underline{t}$ranscription $\underline{r}$egulator (PMTR) has cystein sequence homology at metal binding domain to CueR (copper resistance) protein, which conserves two cysteins (Cys 112 and Cys 120 in PMTR). Gel shift assay revealed that PMTR protein bound to promoter region of Escherichia coli copA (copper-translocating P-type ATPase) and Proteus mirabilis atpase (putative copper-translocating P-type ATPase) genes except that of E. coli zntA (zinc-translocating P-type ATPase) gene. DNase I protection experiment indicated that PMTR protein protected the region over -35 box and close to -10 box. DNase I hypersensitive bases were shown at C and A bases of labeled template strand and at G and C bases of labeled non-template strand of DNA. These hypersensitive bases were appeared in other metalloregulatory proteins of MerR family, which suggests protein-induced DNA bending.

락토스 오페론에서 Cyclic AMP Receptor Protein에 의한 두 결합 부위(CRP1과 CRP2)의 결합 특성에 관한 연구

강종백,권건,Kang, Jong-Baek,Kwon, Gun 한국생명과학회 2003 생명과학회지 Vol.13 No.5

Lactose operon contains two CRP binding sites at promoter(CRP1 site) and operator(CRP2 site) regions at lac operon. CRP protein can bind to both sites with the different binding affinity. CRP1 site, major CRP binding site, acts the transcription activation with the fully unknown mechanism by binding of CRP. In this study, the binding affinities of CRP1 site and CRP2 site were measured with the fluorescein-labeled oligomers, which contain CRP1 site and the three different spacing sequences between GTGA and TCAC at CRP2 site. Results showed that CRP:cAMP complex bound to CRP1 site 3 times more strongly than CRP2 site and the base spacing between GTGA and TCAC was not the only factor to affect the binding affinity of CRP to CRP2 site.

높은 cAMP 농도에서 cAMP 수용성 단백질의 열 안정화

강종백,최영,Kang, Jong-Baek,Choi, Young 한국생명과학회 2003 생명과학회지 Vol.13 No.5

Cyclic AMP receptor proteins(CRP) activate many genes in Escherichia coli by binding of cAMP with not fully known mechanism. CRP existed as apo-CRP in the absence of cAMP, $CRP;(cAMP)_2$$_2$ at low(micromolar) cAMP concentration, or $CRP;(cAMP)_4$ at high(millimolar) concentration of cAMP. This study is designed to measure the thermal stability of S83G CRP, which substituted glycine for serine at amino acid 83 position, with CD spectrapolarimeter at 222nm by the constant elevation of temperature from $20^{\circ]C\; to\; 90^{\circ}C\; at\; 1^{\circ}C/min$. The non-linear regression analysis showed that melting temperatures were 68.4, 72.0, and $82.3^{\circ}C$ for no cAMP, 0.1mM cAMP, and 5mM cAMP, respectively. Result showed the strong thermal stability of CRP by binding of additional cAMP molecules to region between the hinge region and helix-turn-helix(HTH) motif at 5mM cAMP concentration.

산화 그래핀 플랫폼을 이용한 DNA 중합효소의 실시간 형광에세이

강종백 ( Gang Jong Back ) 한국미생물생명공학회(구 한국산업미생물학회) 2013 한국미생물·생명공학회지 Vol.41 No.4

단일가닥 DNA와 이중가닥 DNA의 흡착 율의 차이를 이용하여, 본 연구는 산화 그래핀에 흡착된 단일 가닥 DNA을 사용하여 Klenow fragment의 효소 활성을 검출하기 위하여 실시간 형광에세이 방법을 사용했다. 실험 결과에 의하면, 산화그래핀에 흡착된 형광표지 ssDNA는 형광이 칭(quenching) 되지만, cDNA 첨가에 의해서 흡착된 단일가닥 DNA가 유리되었다. Klenow fragment의 활성을 측정하기 위해서 형광표지 틀(template) DNA, 산화그래핀과 시발체(primer)가 존재할 때, 고분자 반응이 진행됨에 따라 칭된 형광세기가 증가하였다. 그리고 겔 전기영동 실험은 산화 그래핀에서 DNA 합성과 hybridization 반응을 확인하였다. Using the different adsorption properties of ssDNA and dsDNA to GO, this study used a real time and efficient fluorescence assay to detect the enzymatic activity of the Klenow fragment with the adsorbed DNA to GO. Results showed that adsorption of fluorescein-tagged ssDNA to GO resulted in fluorescence quenching and DNA was released from GO by adding complementary DNA. In addition, fluorescence restoration was increased through a polymerization reaction by the Klenow fragment in the presence of a fluorescein-attached template, GO, and primer. Gel electrophoresis was conducted to confirm the hybridization and DNA polymerization reactions on GO.

Trichoderma harzianum 유래 ß-mannanase 에 의한 Locust Bean Gum Galactomannan 가수분해 올리고당의 중합도별 Bifidobacterium spp.에 대한 생육활성

박성은,이희정,김순옥,강종백,박귀근 한국산업식품공학회 2007 산업 식품공학 Vol.11 No.4

Double-Stranded DNA-Templated Copper Nanoclusters for Detection of DNA Polymerase Activity

김정은,강종백 대한화학회 2021 Bulletin of the Korean Chemical Society Vol.42 No.1

DNA polymerase plays an important role in the proliferation and survival of all forms of life and the variety of biotechnological applications. In this study, DNA-templated copper nanoclusters were utilized to detect Klenow fragment exo- (KF-) activity and monitor its inhibition in the presence of 5-fluorouracil by measurement of fluorescence intensities. This study showed that the fluorescence intensities increased continuously for KF- concentrations ranging from 0 to 5 U/mL. In addition, inhibition of KF- activity was measured at 5-fluorouracil concentrations ranging from 0 to 150??M. Using nonlinear regression, the corresponding IC50 was determined to be 6.23??M in the presence of 5.0 U/mL KF-.

Stability and Structure of S128A Mutant cAMP Receptor Protein

최영,강종백 조선대학교 기초과학연구원 2011 조선자연과학논문집 Vol.4 No.3

Cyclic AMP receptor protein(CRP) is involved in the activation of many genes corresponding to catabolite enzymes in Escherichia coli. In this study, mutant CRP(S128A) was used to elucidate the effect of Ser 128 on the cAMP-induced structural change. Based on the protease digestion and thermal analysis, serine 128 in CRP affects the cAMP binding capability and then structural change of CRP protein. In addition, CD spectra in near UV region revealed that S128A CRP retained the sensitive conformation to thermal effect relative to that of wild-type CRP, in spite of identical Tm values in the absence of cAMP.