Investigation of the Substrate Recognition Characteristics and Kinetics of Mammalian Mitochondrial DNA Topoisomerase I

Topoisomerases are DNA-modifying enzymes found in prokaryotes, eukaryotes, viruses and organelles such as chloroplast and mitochondria. Information about these enzymes in eukaryotic systems is mostly limited to nuclear enzymes, although our laboratory has been characterizing the biochemical and biophysical properties of the mammalian mitochondrial topoisomerases. We have determined the polarity of the attachment of mitochondrial topoisomerase I to its substrate DNA. To study the substrate preference and kinetic parameters of mitochondrial topoisomerase I, selected regions of mammalian mitochondrial DNA (mtDNA) were inserted into pGEM plasmid vectors following a series of modification and optimization experiments of currently available methods for PCR-cloning. These mtDNA containing recombinant plasmids were used in a kinetic analysis of the highly purified enzyme. Recombinant plasmids containing the bovine mtDNA heavy and light strand origins of replication (pZT-Hori and pZT-Lori, respectively), a major transcription termination region (pZT-Term) and a portion of cytochrome b gene (pZT-Cytb) were prepared. Two other recombinant plasmids, containing non-mitochondrial DNA inserts (pZT-800 and pZT-400) served as control substrates. Southern hybridization using probes specific for either control or mtDNA-containing plasmids indicated a relative preference by the mitochondrial topoisomerase I to relax supercoils in pZT-Hori and pZT-Term. Quantitative determination of kinetic parameters derived from double-reciprocal Lineweaver-Burk plots showed that recombinant plasmids containing the heavy and light strand origins and the transcription termination region were preferentially relaxed by the mitochondrial enzyme with Km values 2.3 to 3.3-fold lower than controls. The Km values for pZT-Hori, pZT-Lori and pZT-Term were 21.0 +/- 0.9 μΜ, 25.2 +/- 1.0 μM and 17.0 +/- 0.8 μΜ respectively, while those for control plasmids were 57.5 +/- 2.1 μΜ and 56.3 +/- 2.3 μΜ. pZT-Cytb was not preferentially relaxed compared to the control plasmid (Km= 53.4 +/- 2.0 μΜ vs.56.3 +/- 2.3 μΜ respectively) indicating that mitochondrial topoisomerase I preferentially interacts with certain mtDNA sequences but not others. Identical experiments with the purified nuclear enzyme did not differentiate between control or mtDNA containing plasmids

Volume CXX, Number 6, October 25, 2002

3rd Asian Pacific Regional Meeting of the International-Society-for-the-Study-of-Xenobiotics -- MAY 10-12, 2009 -- Bangkok, THAILANDWOS: 000269483300069…Int Soc Study Xenobio

Selected compounds that interfere with the catalytic cycles of type I and type II DNA topoisomerases

4th Asia Pacific Meeting of the International-Society-for-the-Study-of -Xenobiotics (ISSX) -- APR 22-25, 2011 -- Tainan, TAIWANWOS: 000289629400092Int Soc Study Xenobio

Recombination Can either Help Maintain Very Short Telomeres or Generate Longer Telomeres in Yeast Cells with Weak Telomerase Activity▿

Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles

Comparative Cytochrome P450-1A1, -2A6, -2B6, -2C, -2D6, -2E1, -3A5 and -4B1 expressions in human larynx tissue analysed at mRNA level

WOS: 000242307900001PubMed ID: 16894644The metabolic activation of numerous exogenous and endogenous chemicals is catalysed by cytochrome P450 enzymes (CYPs). The aim of this study was to analyse the expression of the individual forms of CYP at the mRNA level in human larynx and quantitatively to compare their expressions in human liver, the main in organ of CYP expression. Individual forms of CYP mRNAs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primers for the CYPs -1A1, -1A2, -2A6,-2B6,-2C, -2D6, -2E1, -3A3/4 ,-3A5, -3A7 and 4B1. An RNA competitor of known copy number, covering the primer sequences necessary to amplify the entire object CYPs within a single molecule, was used as reference. This study reports a consistent detection of mRNAs for the CYPs -1A1, -2A6, -2B6, -2C, -2D6, -2E1, -3A5 and -4B1 in the human larynx tissue. The data indicate that the human larynx highly resembles the lung tissue in CYP content, as a comparable subset of CYP mRNAs was detected in the larynx previously reported for human lung with the exception of CYP1A2. The results are discussed in quantitative ratios of the detected CYP mRNAs in relation to the hepatic CYP expression. Copyright (c) 2006 John Wiley & Sons, Ltd

1H-benzimidazole derivatives as mammalian DNA topoisomerase I inhibitors

WOS: 000249819500011PubMed ID: 17823665Benzimidazole is one of the most important heterocyclic groups manifesting various biological properties, such as antibacterial, antifungal, antimicrobial, antiprotozoal and antihelmintic activities. Several benzimidazole derivatives are also active as inhibitors of type I DNA topoisomerases. In this study, three 1H-benzimidazole derivatives with different electronic characteristics at position 5-, namely 5-chloro-4-(1H-benzimidazole-2-yl)phenoI (Cpd I), 5-methyl-4-(1H-benzimidazole-2-yl)phenol (Cpd II) and 4-(1H-benzimidazole-2-yl)phenol (Cpd III), were synthesized and evaluated for their effects on mammalian type I DNA topoisomerase activity using quantitative in vitro plasmid supercoil relaxation assays. For the structure elucidation of the compounds, melting points, UV, IR, H-1 NMR, C-13 NMR, mass spectral data and elemental analyses were interpreted. Among the compounds, 5-methyl-4-(1H-benzimidazole-2-yl)phenoI (Cpd II) manifested relatively potent topoisomerase I inhibition