scholarly journals Hydroxyl Radicals Are Involved in Cell Killing by the Bacterial Topoisomerase I Cleavage Complex

2009 ◽  
Vol 191 (16) ◽  
pp. 5315-5319 ◽  
Author(s):  
I-Fen Liu ◽  
Thirunavukkarasu Annamalai ◽  
Jeanette H. Sutherland ◽  
Yuk-Ching Tse-Dinh
Keyword(s):  
Escherichia Coli ◽  
Oxidative Damage ◽  
Hydroxyl Radicals ◽  
Topoisomerase I ◽  
Cluster Formation ◽  
Cell Killing ◽  
Cleavage Complex ◽  
Dna Complex

ABSTRACT Escherichia coli expressing SOS-inducing mutant topoisomerase I was utilized to demonstrate that covalent protein-DNA complex accumulation results in oxidative damage. Hydroxyl radicals were detected following mutant topoisomerase induction. The presence of the Fe2+ chelator 2,2′-dipyridyl and an iscS mutation affecting Fe-S cluster formation protect against topoisomerase I cleavage complex-mediated cell killing.

scholarly journals Analysis of RuvABC and RecG Involvement in the Escherichia coli Response to the Covalent Topoisomerase-DNA Complex

2010 ◽  
Vol 192 (17) ◽  
pp. 4445-4451 ◽  
Author(s):  
Jeanette H. Sutherland ◽  
Yuk-Ching Tse-Dinh
Keyword(s):  
Escherichia Coli ◽  
Homologous Recombination ◽  
Dna Cleavage ◽  
Topoisomerase I ◽  
Replication Fork ◽  
Double Strand Breaks ◽  
Strand Breaks ◽  
Sos Induction ◽  
Cleavage Complex ◽  
Dna Complex

ABSTRACT Topoisomerases form a covalent enzyme-DNA intermediate after initial DNA cleavage. Trapping of the cleavage complex formed by type IIA topoisomerases initiates the bactericidal action of fluoroquinolones. It should be possible also to identify novel antibacterial lead compounds that act with a similar mechanism on type IA bacterial topoisomerases. The cellular response and repair pathways for trapped topoisomerase complexes remain to be fully elucidated. The RuvAB and RecG proteins could play a role in the conversion of the initial protein-DNA complex to double-strand breaks and also in the resolution of the Holliday junction during homologous recombination. Escherichia coli strains with ruvA and recG mutations are found to have increased sensitivity to low levels of norfloxacin treatment, but the mutations had more pronounced effects on survival following the accumulation of covalent complexes formed by mutant topoisomerase I defective in DNA religation. Covalent topoisomerase I and DNA gyrase complexes are converted into double-strand breaks for SOS induction by the RecBCD pathway. SOS induction following topoisomerase I complex accumulation is significantly lower in the ruvA and recG mutants than in the wild-type background, suggesting that RuvAB and RecG may play a role in converting the initial single-strand DNA-protein cleavage complex into a double-strand break prior to repair by homologous recombination. The use of a ruvB mutant proficient in homologous recombination but not in replication fork reversal demonstrated that the replication fork reversal function of RuvAB is required for SOS induction by the covalent complex formed by topoisomerase I.

scholarly journals SOS Induction by Stabilized Topoisomerase IA Cleavage Complex Occurs via the RecBCD Pathway

2008 ◽  
Vol 190 (9) ◽  
pp. 3399-3403 ◽  
Author(s):  
Jeanette H. Sutherland ◽  
Bokun Cheng ◽  
I-Fen Liu ◽  
Yuk-Ching Tse-Dinh
Keyword(s):  
Escherichia Coli ◽  
Cell Death ◽  
Topoisomerase I ◽  
Single Stranded Dna ◽  
Sos Induction ◽  
Cleavage Complex

ABSTRACT Accumulation of mutant topoisomerase I cleavage complex can lead to SOS induction and cell death in Escherichia coli. The single-stranded break associated with mutant topoisomerase I cleavage complex is converted to double-stranded break, which then is processed by the RecBCD pathway, followed by association of RecA with the single-stranded DNA.

scholarly journals Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases

2002 ◽  
Vol 46 (6) ◽  
pp. 1785-1792 ◽  
Author(s):  
Shabana I. Khan ◽  
Alison C. Nimrod ◽  
Mohammed Mehrpooya ◽  
John L. Nitiss ◽  
Larry A. Walker ◽  
...  
Keyword(s):  
Antifungal Activity ◽  
Mode Of Action ◽  
Mammalian Cells ◽  
Topoisomerase I ◽  
Topoisomerase Ii ◽  
Cell Killing ◽  
In Vitro Assays ◽  
Fungal Cell ◽  
Cleavage Complex

ABSTRACT The azafluoranthene alkaloid eupolauridine has previously been shown to have in vitro antifungal activity and selective inhibition of fungal topoisomerase I. The present study was undertaken to examine further its selectivity and mode of action. Eupolauridine completely inhibits the DNA relaxation activity of purified fungal topoisomerase I at 50 μg/ml, but it does not stabilize the cleavage complex of either human or fungal topoisomerase I. Cleavage complex stabilization is the mode of action of topoisomerase I targeting drugs of the camptothecin family. Also, unlike camptothecin, eupolauridine does not cause significant cytotoxicity in mammalian cells. To determine if the inhibition of topoisomerase I is the principal mode of antifungal action of eupolauridine, Saccharomyces cerevisiae strains with alterations in topoisomerase genes were used in clonogenic assays. The antifungal activity of eupolauridine was not diminished in the absence of topoisomerase I; rather, the cells lacking the enzyme were more sensitive to the drug. Cell-killing activity of eupolauridine was also more pronounced in cells that overexpressed topoisomerase II. In vitro assays with the purified yeast enzyme confirmed that eupolauridine stabilized topoisomerase II covalent complexes. These results indicate that a major target for fungal cell killing by eupolauridine is DNA topoisomerase II rather than topoisomerase I, but does not exclude the possibility that the drug also acts against other targets.

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