• BMB Reports
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• 제36권6호
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• pp.525-528
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• 2003

The role of 3' exonuclease excision in DNA polymerization was evaluated in primer extensions using 3' allele-specific primers that had exonuclease-digestible and exonuclease-resistant 3' termini. With exonuclease-digestible unmodified 3' mismatched primers, the exo+ polymerase yielded template-dependent products. Using exonuclease-resistant 3' mismatched primers, no primer-extended product resulted from exo+ polymerase. As a control, polymerase without proofreading activity yielded primer-dependent products from 3' mismatched primers. These data indicated that a successful removal of the mismatch is required for DNA polymerization from the 3' mismatched primers by exo+ polymerase. In addition to the well-known proofreading from this mismatch removal, the premature termination in DNA polymerization, due to the failure of the efficient removal of the mismatched nucleotides, worked as an off-switch in maintaining the high fidelity in DNA replication from exo+ polymerase.

• BMB Reports
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• 제37권3호
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• pp.269-274
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• 2004

DNA polymerases without the 3' exonuclease function ($exo^-$ pol) have been widely used in sequencing and SNP genotyping. As a major player that expedited the coming of the postgenomic era, $exo^-$ polymerases worked remarkably well in the Human Genome Sequencing Project. However, it has become a challenge for this class of polymerases to efficiently screen the large number of SNPs that are found in the human genome. For more than three decades it has been recognized that polymerase fidelity varied according to the presence of proofreading activity that is mediated by its internal 3' exonuclease. Polymerases with proofreading function are famous for their high fidelity in DNA replication both in vivo and in vitro, but this well-known class of polymerases has been almost completely neglected in genetic analysis in the postgenomic era. We speculate that $exo^+$ polymerases may exhibit higher nucleotide identification ability when compared to $exo^-$ polymerases for an in vitro genetic analysis. With the application of $exo^+$ polymerases in SNP assays, a novel mechanism for the maintenance of DNA replication, the on/off switch, was discovered. Two new SNP assays have been developed to carry out genome-wide genotyping, taking advantage of the enzymatic properties of $exo^+$ polymerases. Furthermore, the on/off switch mechanism embodies a powerful nucleotide identification ability, which can be used to discriminate the bases that are upstream of the 3' terminus, and thus defines a new concept in de novo sequencing technology. Application of $exo^+$ polymerases to genetic analysis, and especially SNP assays, will greatly accelerate the pace to personalized medicine.

• BMB Reports
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• 제36권6호
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• pp.529-532
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• 2003

The potential physiological role and technological application of the premature termination of DNA polymerization through the off-switch of exo+ polymerases were studied using 3' phosphorothioate-modified or unmodified primers with single base mismatch distal to the 3' terminus. With exonuclease-digestible unmodified primers, a gradient premature termination of DNA polymerization was observed when amplified with exo+ polymerases. With 3' allele specific phosphorothioate-modified primers, an efficient off-switch effect occurred in the discrimination of a single nucleotide polymorphism when directly using genomic DNA. Clearly, the off-switch of exo+ polymerases is useful in biomedical research.

• BMB Reports
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• 제38권1호
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• pp.24-27
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• 2005

The role of 3' exonuclease excision in DNA polymerization was evaluated for primer extension using inert allele specific primers with exonuclease-digestible ddNMP at their 3' termini. Efficient primer extension was observed in amplicons where the inert allele specific primers and their corresponding templates were mismatched. However, no primer-extended products were yielded by matched amplicons with inert primers. As a control, polymerase without proofreading activity failed to yield primer extended products from inert primers regardless of whether the primers and templates were matched or mismatched. These data indicated that activation was undertaken for the inert allele specific primers through mismatch proofreading. Complementary to our previously developed SNP-operated on/off switch, in which DNA polymerization only occurs in matched amplicon, this new mutation detection assay mediated by $exo^+$ DNA polymerases has immediate applications in SNP analysis independently or in combination of the two assays.

• Journal of Microbiology and Biotechnology
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• 제32권1호
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• pp.91-98
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• 2022

Tetanus is a potentially fatal public health illness resulted from the neurotoxins generated by Clostridium tetani. C. tetani is not easily culturable and culturing the relevant bacteria from infected wounds has rarely been useful in diagnosis; PCR-based assays can only be conducted at highly sophisticated laboratories. Therefore, a real-time recombinase polymerase amplification assay (Exo-RPA) was constructed to identify the fragments of the neurotoxin gene of C. tetani. Primers and the exo probe targeting the conserved region were designed, and the resulting amplicons could be detected in less than 20 min, with a detection limit of 20 copies/reaction. The RPA assay displayed good selectivity, and there were no cross-reactions with other infectious bacteria common in penetrating wounds. Tests of target-spiked serum and pus extract revealed that RPA is robust to interfering factors and has great potential for further development for biological sample analysis. This method has been confirmed to be reliable for discriminating between toxic and nontoxic C. tetani strains. The RPA assay dramatically improves the diagnostic efficacy with simplified device architecture and is a promising alternative to real-time PCR for tetanus detection.

• Journal of Microbiology and Biotechnology
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• 제8권5호
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• pp.471-477
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• 1998

Taq DNA polymerase from Thermus aquaticus is very useful in the polymerase chain reaction. Taq DNA polymerase is classified in the pol I family, represented by E. coli DNA polymerase I. The three-dimensional structural alignment of 3'-5'exonuclease domains from the pol I family DNA polymerases explains why Taq DNA polymerase does not carry out proofreading in polymerase chain reactions. Three sequence motifs, Exo I, II, and III, must exist to carry out 3'-5'exonuclease activity for proof- reading by a 3'-5'exonuclease reaction, but these are abolished in Taq DNA polymerase. The key catalytic module in 3'-5'exonuclease is two metal ions chelated by four active-site carboxylic amino acids. Taq DNA polymerase was mutagenized to construct the catalytic module in the active site. The circular dichroism technique supported the formation of the catalytic module, and the radioactive assay showed that the 3'-5'exonuclease activity doubled in the mutant Taq DNA polymerase.

• Journal of Microbiology and Biotechnology
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• 제33권4호
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• pp.430-440
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• 2023

The type three secretion system (T3SS) is a major virulence system of Pseudomonas aeruginosa (P. aeruginosa). The effector protein Exotoxin S (ExoS) produced by P. aeruginosa is secreted into the host cells via the T3SS. For the purpose of an experiment on inhibitors with regard to ExoS secretion, we developed a sandwich-type enzyme-linked immunosorbent assay (ELISA) system. Quercetin was selected because it has a prominent ExoS inhibition effect and also is known to have anti-inflammatory and antioxidant effects on mammalian cells. In this study, we investigated the effects of quercetin on the expression and secretion of ExoS using ELISA and Western blot analysis methods. The results showed that the secretion of ExoS was significantly decreased by 10 and 20 µM of quercetin. Also, popB, popD, pscF, and pcrV which are composed of the T3SS needle, are reduced by quercetin at the mRNA level. We also confirmed the inhibitory effect of quercetin on cytokines (IL-6, IL-1β, and IL-18) in P. aeruginosa-infected H292 cells by real-time polymerase chain reaction (PCR) and ELISA. Collectively, quercetin inhibits the secretion of ExoS by reducing both ExoS production and the expression of the needle protein of T3SS. Furthermore, these results suggest that quercetin has the potential to be used as an anti-toxic treatment for the inflammatory disease caused by P. aeruginosa infection.

• 한국미생물·생명공학회지
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• 제24권4호
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• pp.415-422
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• 1996

A gene coding for a pyruvyl transferase enzyme involved in exopolysaccharide biosynthesis of Zoogloea ramigera 115SLR was isolated and sequenced. A 4.5 kb of BamHI DNA fragment was isolated from chromosomal DNA using a probe derived from ketal pyruvyl transferase gene of Xanthomonas campestris. The nucleotide sequence of 2.66 kb Pst1/HindIII DNA fragment which was homology with a probe revealed the existence of two complete open reading frames (ORF2 and ORF3) and two partial open reading frames (ORFI and ORF4). The deduced amino acid sequence of ORF3 was homologous to the ketalase (GumL product) of X campestris with 49.5% of similarity and 21.6% of identity. ORF2 on the other hand showed the higher identity with the ketalase (ExoV product) of Rhizobium meliloti (36%) as well as the ketalase of X campestris (23%) than that of ORF3. A gene product of ORF2 was determined with a bacteriophage T7 RNA polymerase/promoter system in E. coli. The molecular weight of protein was 33,500 dalton.

• Molecules and Cells
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• 제42권9호
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• pp.637-645
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• 2019

Effector-triggered immunity (ETI) is an effective layer of plant defense initiated upon recognition of avirulence (Avr) effectors from pathogens by cognate plant disease resistance (R) proteins. In rice, a large number of R genes have been characterized from various cultivars and have greatly contributed to breeding programs to improve resistance against the rice blast pathogen Magnaporthe oryzae. The extreme diversity of R gene repertoires is thought to be a result of co-evolutionary history between rice and its pathogens including M. oryzae. Here we show that Pii is an allele of Pi5 by DNA sequence characterization and complementation analysis. Pii-1 and Pii-2 cDNAs were cloned by reverse transcription polymerase chain reaction from the Pii-carrying cultivar Fujisaka5. The complementation test in susceptible rice cultivar Dongjin demonstrated that the rice blast resistance mediated by Pii, similar to Pi5, requires the presence of two nucleotide-binding leucine-rich repeat genes, Pii-1 and Pii-2. Consistent with our hypothesis that Pi5 and Pii are functionally indistinguishable, the replacement of Pii-1 by Pi5-1 and Pii-2 by Pi5-2, respectively, does not change the level of disease resistance to M. oryzae carrying AVR-Pii. Surprisingly, Exo70F3, required for Pii-mediated resistance, is dispensable for Pi5-mediated resistance. Based on our results, despite similarities observed between Pi5 and Pii, we hypothesize that Pi5 and Pii pairs require partially distinct mechanisms to function.