• Korean Journal of Microbiology
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• v.32 no.3
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• pp.208-214
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• 1994

We screened many species from a wide variety of bacterial genera for a new type II restriction endonuclease. The purification and characterization of SolI from a soil isolate, Streptoverticillium olivoverticillatum are described here. The enzyme turned out to be an isoschizomer of BamHI. It recognized the hexanucleotide sequence of 5'-G$\downarrow$GATCC-3' and cleaved as in dicated by the arrow, generating a 4 base 5' extension. Unlike its isoschizomer, BamHI, the activity was sensitive to dam methylation within the recognition sequence. Following ammonium sulfate fractionation of the crude extract, heparin-agarose and Affi-gel Blue column chromatography were employed to purify the enzyme. SolI required at least 0.2 mM of $MgCl_2$ for the cleavage to occur. The enzyme exhibited its maximal activity in the absence of NaCl, but was inhibited completely in the presence of 120 mM NaCl. The pH and temperature optima for activity were pH 8.6 and $40^{\circ}C$, respectively. The molecular weight of SolI was estimated to be 43,000 Da by Superose-12 gel filtraion chromatography.

• Journal of the Korean Magnetic Resonance Society
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• v.24 no.3
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• pp.70-76
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• 2020

The CRISPR-Cas system provides an adaptive immunity for bacteria and archaea against invading phages or foreign plasmids. In the type II CRISPR-Cas system, a single effector protein Cas9 and a guide RNA form an RNA-guided endonuclease complex that can degrade DNA targets of foreign origin. To avoid the Cas9-mediated destruction, phages evolved anti-CRISPR (Acr) proteins that neutralize the host bacterial immunity by inactivating the CRISPR-Cas system. Here we report the backbone ¹H, ¹⁵N, and ¹³C resonance assignments of AcrIIA5 that inhibits the endonuclease activity of type II-A Streptococcus thermophilus Cas9 and also Streptococcus pyogenesis Cas9 using triple resonance nuclear magnetic resonance spectroscopy. The backbone chemical shifts of AcrIIA5 predict a disordered region at the N-terminus, followed by an αββββαβββ fold.

• Journal of the Korean Magnetic Resonance Society
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• v.22 no.3
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• pp.71-75
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• 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.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1995.04a
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• pp.80-80
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• 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.

• BMB Reports
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• v.36 no.5
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• pp.520-523
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• 2003

The proliferation and differentiation signals of myelogeneous U937 cells are provided by extracellular stimuli, such as lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA). In a DNA-native-polyacrylamide gel assay system, we demonstrated that a particular nuclease activity is expressed in PMA-stimulated U937 cells and secreted into the culture medium. The nuclease activity was induced in U937 cells by LPS treatment, while the secretion of the enzyme was undetected in the culture medium. Therefore, it is likely that the expression and secretion of the particular nuclease in U937 cells are controlled by extracellular stimulations, such as PMA and LPS treatment.

• Microbiology and Biotechnology Letters
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• v.14 no.2
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• pp.161-168
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• 1986

A 1.95Kb Sau3Al fragment coding for $\alpha$-amylase from Bacillus amyloliquefaciens was isolated by the shotgun method using Escherichia coli as a host. The genome of Bacillus amyloliquefaciens was partially digested with the restriction endonuclease Sau3Al and joined to plasmid YRp7 cleaved with the restriction endonuclease BamHI. The $\alpha$-amylase gene present in a 1.95Kb insert was stably maintained and expressed in Escherichia coli. The amount of $\alpha$-amylase activity produced by Escherichia coli containing the hybrid plasmid pEA24 was about 65% of the activity produced by the donor Bacillus amyloliquefaciens strain. The properties of $\alpha$-amylase produced by Escherichia coli were very similar to those produced by Bacillus amyloliquefaciens as based on optimum temperature, pH, and effect of CaCl$_2$ concentration. About 70% of the $\alpha$-amylase produced by Escherichia coli was localized in the periplasmic space, whereas the remaining enzyme was localized in the inner part of the cell.

• Proceedings of the Korea Crystallographic Association Conference
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• 2003.05a
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• pp.16-16
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• 2003

Mismatches in a DNA duplex are mainly due to DNA duplication errors that are generated by improper function of DNA polymerase. MutS, MutL and MutH are crucial proteins for the initiation of the methyl-directed mismatch repairing in bacteria. MutS has an ATPase activity md recognize the mismatched or unpaired bases on DNA. After binding to a mismatch, MutS recruits MutL to mediate the activation of MutH an endonuclease, which cleaves the 5' site of d(GATC) on the un-methylated strand. Both MutL and MutS also have essential roles in the subsequent removal and re-synthesis of the daughter strand. We have determined the crystal structures of either intact or active fragments of each of these proteins, both alone and complexed with ligands (DNA, ADP and ATP). The biochemical and mutagenesis studies based on the detailed 3-D structures led to new insights into the role of the ATPase activity of MutS in the mismatch recognition and directions for future investigation of mismatch repair.

• Korean Journal of Microbiology
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• v.27 no.3
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• pp.188-193
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• 1989

Gene for lactose catabolism in Lactobacillus casei SW-M1 was encoded by a 60Kb metabolic plasmid. A derivative of only 10kb, pPlac 15 of recombinant plasmid, was constructed by introducing into pBR322 and was cloned into E. coli using restriction endonuclease Pst I. A 10kb insery DNA in plasmid pBR322 was identified as a gene encoded phospho-$\beta$-galactosidase by the determination of enzyme activity. Phospho-$\beta$-galactosidase was apparently expressed in E. coli. The enzyme activities of cell-free extract from transformant E. coli HB101 carrying pPLac 15 DNA were not different from that of L. casei as a donor strain on the basis of enzyme properites. However, specific activity of phospho-$\beta$-galactosidase in the cloned strain with Lac $Y^{-}$ phenotype of E. coli HB101 was lower than that in L. casei strain.

• The Plant Pathology Journal
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• v.25 no.1
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• pp.47-53
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• 2009

Sequence analysis of the Chlorella virus SS-2 revealed one putative nuclease gene that is 807 bp long and encodes a 31kDa protein. Multiple sequence alignment analysis reveals the presence of highly conserved PD-(D/E)XK residues in the encoded protein. The gene cloned into an expression vector was expressed as a His-tagged fusion protein in chaperone containing pKJE7 cells. The recombinant protein was purified using a His-Trap chelating HP column and used for functional analysis. Exonuclease activity of the SS-2 nuclease was detected when the DNA substrates, such as linear ssDNA, PCR amplicon, linear dsDNA with 5'-overhang ends, 3'-overhang ends, or blunt ends were used. Covalently closed circular DNA was also degraded by the SS-2 recombinant protein, suggesting that the SS-2 nuclease has an endonuclease activity. Stable activity of SS-2 nuclease was observed between $10^{\circ}C$ and $50^{\circ}C$. The optimum pH concentrations for the SS-2 nuclease were pH 6.0-8.5. Divalent ions inhibited the SS-2 nuclease activity.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.301-305
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• 1994

In catalytic properties of the restriction enonuclease, SdiI, which was purified from Streptomyces diastatochromogenes, this enzyme was active at wide range between pH 7.0 and 12.5, and up to $60^{\circ}C$ and 500 mM of NaCl concentration. It was stable between 20^{\circ}C$ and $60^{\circ}C$, and essentially requires $MgCl_2$ for endonuclease activity. The restriction map of lambda DNA which was obtained by double digestion with various enzymes suggested SdiI to be an isoschizomer of XhoI. From the determination of restriction site based on DNA sequencing method, recognition and cleavage specificity of SdiI was concluded as: 5‘-C${\downarrow}$TCGA G-3' 3'-G AGCT${\uparrow}$C-5'