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Journal ArticleDOI

In the Presence of Subunit A Inhibitors DNA Gyrase Cleaves DNA Fragments as Short as 20 bp at Specific Sites

01 Feb 1997-Nucleic Acids Research (Oxford University Press)-Vol. 25, Iss: 3, pp 604-610
TL;DR: It is proposed that subunit A inhibitors interact with DNA at inhibitor-specific positions, thus determining cleavage sites by forming ternary complexes between DNA, inhibitors and DNA gyrase.
Abstract: A key step in the supercoiling reaction is the DNA gyrase-mediated cleavage and religation step of double-stranded DNA. Footprinting studies suggest that the DNA gyrase binding site is 100-150 bp long and that the DNA is wrapped around the enzyme with the cleavage site located near the center of the fragment. Subunit A inhibitors interrupt this cleavage and resealing cycle and result in cleavage occurring at preferred sites. We have been able to show that even a 30 bp DNA fragment containing a 20 bp preferred cleavage sequence from the pBR322 plasmid was a substrate for the DNA gyrase-mediated cleavage reaction in the presence of inhibitors. This DNA fragment was cleaved, although with reduced efficiency, at the same sites as a 122 bp DNA fragment. A 20 bp DNA fragment was cleaved with low efficiency at one of these sites and a 10 bp DNA fragment was no longer a substrate. We therefore propose that subunit A inhibitors interact with DNA at inhibitor-specific positions, thus determining cleavage sites by forming ternary complexes between DNA, inhibitors and DNA gyrase.

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Citations
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Journal ArticleDOI
TL;DR: In this paper, the authors show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmis gyrase (Msgyrase) in the absence of fluoroquinolones (FQs), while in their presence, MsMfpa decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs.
Abstract: DNA gyrase, a type II topoisomerase, introduces negative supercoils into DNA using ATP hydrolysis. The highly effective gyrase-targeted drugs, fluoroquinolones (FQs), interrupt gyrase by stabilizing a DNA-cleavage complex, a transient intermediate in the supercoiling cycle, leading to double-stranded DNA breaks. MfpA, a pentapeptide-repeat protein in mycobacteria, protects gyrase from FQs, but its molecular mechanism remains unknown. Here, we show that Mycobacterium smegmatis MfpA (MsMfpA) inhibits negative supercoiling by M. smegmatis gyrase (Msgyrase) in the absence of FQs, while in their presence, MsMfpA decreases FQ-induced DNA cleavage, protecting the enzyme from these drugs. MsMfpA stimulates the ATPase activity of Msgyrase by directly interacting with the ATPase domain (MsGyrB47), which was confirmed through X-ray crystallography of the MsMfpA-MsGyrB47 complex, and mutational analysis, demonstrating that MsMfpA mimics a T (transported) DNA segment. These data reveal the molecular mechanism whereby MfpA modulates the activity of gyrase and may provide a general molecular basis for the action of other pentapeptide-repeat proteins.

8 citations

Journal ArticleDOI
TL;DR: Type II topoisomerases change DNA topology by passage of one DNA duplex through a transient double-stranded break in another (the gate, G-segment), and catenation was observed when one, but not both, of the DNA-DNA duplexes was replaced by a DNA-RNA duplex.

7 citations

Journal ArticleDOI
TL;DR: A workflow consisting of various methods for de novo design and 2D-QSAR model based on molecular fingerprints was constructed to extract the bioactive molecular fingerprints from a data set of DNA-gyrase inhibitors with new structure and mechanism, achieving a potential DNA-Gyrase inhibitor.

7 citations

Journal ArticleDOI
TL;DR: In this paper, cyclocondensation of orthophenylendiamines with 3-bromopropionic acid in PPA afforded 2-vinylbenzimidazoles which were subsequently converted to their corresponding 2-(1,2-dibromoethyl)-1H-benzinidazole on treatment with bromine.

6 citations

Dissertation
01 Jan 1998
TL;DR: Investigating the M echanism and Energy C oupling o f DNA gyrase and the proposed role o f these re s id u es is in n ucleo tide b ind ing, tran s itio n -s ta te s tab ilisa tio n and tr ig g erin g conform ational changes follow ing A TP hydrolysis.
Abstract: Investigating the M echanism and Energy C oupling o f DNA gyrase C lare V ictoria Sm ith DNA gyrase is the bacterial type II topoisom erase w hich couples the free energy o f A TP hydro lysis to the in troduction o f negative superco ils into DNA. The active enzym e is com posed o f tw o G yrA and tw o GyrB subunits form ing an A 2 B 2 com plex. The specific su p erco ilin g ac tiv ity o f G yrB w as found to be consis ten tly low er than the specific supercoiling activ ity o f G yrA and this is believed to be due to m is-folding o f the subunit. E xpression as a th io redox in -fu sion pro tein d id not im prove the specific superco iling activ ity o f G yrB . The C -term inal 47 kD a dom ain o f G yrB (G yrB 47) was over-expressed as a so lub le p ro tein w hen fused to th ioredoxin . T his dom ain in teracts w ith G yrA and D NA . In com plex w ith G yrA , G yrB 47 supports qu ino loneand C a -+-induced D NA cleavage and has A T P-independent relaxation activ ity w hich is com parab le w ith that o f full-length G yrB . G yrB 47 also supports low level A TP-independent decatenation. P ro te in c ro ss lin k in g w as used to in vestiga te nuc leo tid e -, D N A and d ru g -in d u ced confo rm ationa l changes during the reaction cycle o f the enzym e. U pon addition o f the non-hydro lysab le A TP analogue, A DPN P, there w as an increase in crosslinking betw een the G yrB subunits. In the presence o f DNA , crosslinks betw een the G yrA subunits w ere identified. U sing lim ited proteolysis and im m unodetection, these crosslinks were show n to be betw een the N -term inal 64 kD a dom ains o f G yrA . E xam ination o f the X-ray crystal structure o f the 43 kD a dom ain o f GyrB (G yrB 43) show s that the side chains o f G ln335 and Lys337 interact w ith the y-phosphate o f the ATP. Both these residues are highly conserved am ong type II topoisom erases. The proposed role o f these re s id u es is in n ucleo tide b ind ing , tran s itio n -s ta te s tab ilisa tio n and tr ig g erin g conform ational changes follow ing A TP hydrolysis. S ite-directed m utagenesis w as used to convert G ln335 to Asn and A la and Lys337 to Gin and Ala. No clear role for G ln335 has been es tab lish ed in nucleo tide b inding , A TP hydro lysis or triggering con fo rm ationa l changes. H ow ever m utations at Lys337 lead to a m odest decrease in nucleo tide binding and a large reduction in A TP hydrolysis (~10*-fold decrease in ACai)T herefore Lys337 is a critica l residue for A TP turnover and the results are consistent w ith its involvem ent in transition-state stabilisation. TABLE OF CONTENTS Page Acknowledgements ii Abbreviations iii Abstract v Table of contents vi List of figures xi List of tables xv Chapter 1 Introduction 1.

3 citations

References
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Book
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol

215,169 citations

BookDOI
04 Jun 2019
TL;DR: In this paper, the authors present a new book about developments in cancer chemotherapy to read, which they call "Let's read!" They also discuss the importance of reading and the obligation to read.
Abstract: Let's read! We will often find out this sentence everywhere. When still being a kid, mom used to order us to always read, so did the teacher. Some books are fully read in a week and we need the obligation to support reading. What about now? Do you still love reading? Is reading only for you who have obligation? Absolutely not! We here offer you a new book enPDFd developments in cancer chemotherapy to read.

79 citations