The Acidic Triad Conserved in Type IA DNA Topoisomerases Is Required for Binding of Mg(II) and Subsequent Conformational Change

Zhu, Chang-Xi; Tse‐Dinh, Yuk‐Ching

doi:10.1074/jbc.275.8.5318

Cited by 61 publications

(86 citation statements)

References 22 publications

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“…Mg 2ϩ coordination with these residues is essential for enzyme function. The mutations in these acidic residues lead to impairment in the catalytic activity and cytotoxicity (38,39), indicating that targeting the topoI through these residues may impair the enzyme function. In our earlier studies, we showed that the mutations of the individual amino acids, D108 and E112, of the motif in MstopoI to alanine resulted in 20-and 5-fold losses of activity, respectively (38), while mutations of the acidic residues in MttopoI led to complete loss of activity (25).…”

Section: Resultsmentioning

confidence: 99%

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

Godbole

Ahmed

Bhat

et al. 2015

Antimicrob Agents Chemother

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We describe inhibition of Mycobacterium tuberculosis topoisomerase I (MttopoI), an essential mycobacterial enzyme, by two related compounds, imipramine and norclomipramine, of which imipramine is clinically used as an antidepressant. These molecules showed growth inhibition of both Mycobacterium smegmatis and M. tuberculosis cells. The mechanism of action of these two molecules was investigated by analyzing the individual steps of the topoisomerase I (topoI) reaction cycle. The compounds stimulated cleavage, thereby perturbing the cleavage-religation equilibrium. Consequently, these molecules inhibited the growth of the cells overexpressing topoI at a low MIC. Docking of the molecules on the MttopoI model suggested that they bind near the metal binding site of the enzyme. The DNA relaxation activity of the metal binding mutants harboring mutations in the DxDxE motif was differentially affected by the molecules, suggesting that the metal coordinating residues contribute to the interaction of the enzyme with the drug. Taken together, the results highlight the potential of these small molecules, which poison the M. tuberculosis and M. smegmatis topoisomerase I, as leads for the development of improved molecules to combat mycobacterial infections. Moreover, targeting metal coordination in topoisomerases might be a general strategy to develop new lead molecules.

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Section: Resultsmentioning

confidence: 99%

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

Godbole

Ahmed

Bhat

et al. 2015

Antimicrob Agents Chemother

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“…In contrast, mutations at Glu-9 of E. coli topoisomerase I behaved somewhat differently. Replacement by alanine almost completely eliminated DNA cleavage, but glutamine supported high levels of DNA cleavage (although DNA relaxation activity was still severely compromised) (13,44,45). Moreover, replacement by alanine of Asp-111 (equivalent to Spo11p Asp-288) supported high levels of DNA cleavage and relaxation activity, whereas substitution at Asp-113 (equivalent to Asp-290 of Spo11p) caused a roughly 5-to 10-fold decrease in relaxation and a modest defect in DNA cleavage.…”

Section: Figmentioning

confidence: 99%

Identification of Residues in Yeast Spo11p Critical for Meiotic DNA Double-Strand Break Formation

Diaz

Alcid

Berger

et al. 2002

Molecular and Cellular Biology

105

124

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Saccharomyces cerevisiae Spo11 protein (Spo11p) is thought to generate the DNA double-strand breaks (DSBs) that initiate homologous recombination during meiosis. Spo11p is related to a subunit of archaebacterial topoisomerase VI and appears to cleave DNA through a topoisomerase-like transesterase mechanism. In this work, we used the crystal structure of a fragment of topoisomerase VI to model the Spo11p structure and to identify amino acid residues in yeast Spo11p potentially involved in DSB catalysis and/or DNA binding. These residues were mutated to determine which are critical for Spo11p function in vivo. Mutation of Glu-233 or Asp-288, which lie in a conserved structural motif called the Toprim domain, abolished meiotic recombination. These Toprim domain residues have been implicated in binding a metal ion cofactor in topoisomerases and bacterial primases, supporting the idea that DNA cleavage by Spo11p is Mg 2؉ dependent. Mutations at an invariant arginine (Arg-131) within a second conserved structural motif known as the 5Y-CAP domain, as well as three other mutations (E235A, F260R, and D290A), caused marked changes in the DSB pattern at a recombination hotspot, suggesting that Spo11p contributes directly to the choice of DNA cleavage site. Finally, certain DSB-defective mutant alleles generated in this study conferred a semidominant negative phenotype but only when Spo11p activity was partially compromised by the presence of an epitope tag. These results are consistent with a multimeric structure for Spo11p in vivo but may also indicate that the amount of Spo11 protein is not a limiting factor for DSB formation in normal cells.Homologous recombination during meiosis in Saccharomyces cerevisiae proceeds via the formation and subsequent repair of DNA double-strand breaks (DSBs) (reviewed in references 24 and 38). Formation of these DSBs requires the products of at least 10 genes, including SPO11. The Spo11 protein (Spo11p) was found covalently linked to the 5Ј-strand termini of DSBs in certain mutants (e.g., rad50S) that are defective for the normal 5Ј-to-3Ј nucleolytic processing of DSB ends (23), and Spo11p shares sequence similarity with a subunit of an archaebacterial topoisomerase (6). Based on these observations, it is thought that Spo11p is the catalytic subunit of the meiotic DNA cleaving activity and that it cuts DNA by a topoisomerase-like transesterification reaction. The role of Spo11p in promoting meiotic recombination initiation is widely conserved, as functional homologs have been characterized in fungi, plants, and animals (3,10,11,16,18,26,30,31,35,43).It is likely that all of the archaebacterial Spo11p homologs function as type II topoisomerases, but topoisomerase activity has been directly demonstrated only for the enzyme from Sulfolobus shibatae (5,8). Because the amino acid sequence of this topoisomerase is unlike the previously known eukaryotic and prokaryotic type II enzymes, it was named topoisomerase VI to distinguish it from these proteins (6). Topoisomerase VI is an A 2 B 2 heterot...

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“…Two basic residues, Lys-13 and Arg-321 are required for the relaxation activities and have been proposed to interact with the DNA phosphates for DNA cleavage (16 -19). Three acidic residues Asp-111, Asp-113, and Glu-115 have been proposed to be responsible for coordinating two Mg 2ϩ ions (20). Asp-111 and Asp-113 are part of the TOPRIM motif DXDXXG conserved in nucleotidyl transferases for binding of divalent ions required for catalytic activity (21).…”

Section: From the Department Of Biochemistry And Molecular Biology Nmentioning

confidence: 99%

The Strictly Conserved Arg-321 Residue in the Active Site of Escherichia coli Topoisomerase I Plays a Critical Role in DNA Rejoining

Narula

Annamalai

Aedo

et al. 2011

Journal of Biological Chemistry

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The strictly conserved arginine residue proximal to the active site tyrosine of type IA topoisomerases is required for the relaxation of supercoiled DNA and was hypothesized to be required for positioning of the scissile phosphate for DNA cleavage to take place. Mutants of recombinant Yersinia pestis topoisomerase I with hydrophobic substitutions at this position were found in genetic screening to exhibit a dominant lethal phenotype, resulting in drastic loss in Escherichia coli viability when overexpressed. In depth biochemical analysis of E. coli topoisomerase I with the corresponding Arg-321 mutation showed that DNA cleavage can still take place in the absence of this arginine function if Mg 2؉ is present to enhance the interaction of the enzyme with the scissile phosphate. However, DNA rejoining is inhibited in the absence of this conserved arginine, resulting in accumulation of the cleaved covalent intermediate and loss of relaxation activity. These new experimental results demonstrate that catalysis of DNA rejoining by type IA topoisomerases has a more stringent requirement than DNA cleavage. In addition to the divalent metal ions, the side chain of this arginine residue is required for the precise positioning of the phosphotyrosine linkage for nucleophilic attack by the 3-OH end to result in DNA rejoining. Small molecules that can interfere or distort the enzyme-DNA interactions required for DNA rejoining by bacterial type IA topoisomerases could be developed into novel antibacterial drugs.Cellular processes such as replication and transcription require strand separation of duplex DNA, which can potentially lead to excess DNA supercoiling or strand entanglement. DNA topoisomerases are enzymes that overcome the topological barriers in DNA for cellular processes to proceed at optimal rates (1). These enzymes function by transiently cleaving and rejoining DNA through the formation of an intermediate covalent enzyme-DNA complex (1, 2). Topoisomerases are classified into two types based on the number of strands they cleave. Type I topoisomerases cleave a single strand of DNA, whereas type II topoisomerases cleave both strands of DNA. Each type is further subdivided into subgroups that are functionally and structurally dissimilar (1).Type IB and type IIA topoisomerases are well utilized targets for various anticancer and antibacterial drugs used in therapy. These drugs are effective in killing cancer and bacterial cells because they lead to the accumulation of the covalent intermediate formed between topoisomerase and cleaved DNA (3-7). The emergence of bacterial pathogens resistant to all available antibiotics poses a serious global public health problem. Hence, there is an urgent need for the discovery of a new class of antibacterial compounds. The type IA topoisomerase family includes bacterial and archael topoisomerase I, topoisomerase III, and eukaryotic topoisomerase III (8, 9). At least one type IA topoisomerase is present in each bacterial genome sequenced and annotated thus far (10, 11). Hence, type IA...

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The Acidic Triad Conserved in Type IA DNA Topoisomerases Is Required for Binding of Mg(II) and Subsequent Conformational Change

Cited by 61 publications

References 22 publications

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

Targeting Mycobacterium tuberculosis Topoisomerase I by Small-Molecule Inhibitors

Identification of Residues in Yeast Spo11p Critical for Meiotic DNA Double-Strand Break Formation

The Strictly Conserved Arg-321 Residue in the Active Site of Escherichia coli Topoisomerase I Plays a Critical Role in DNA Rejoining

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