Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Holliday junction resolvases (HJRs) are key enzymes of DNA recombination. A detailed computer analysis of the structural and evolutionary relationships of HJRs and related nucleases suggests that the HJR function has evolved independently from at least four distinct structural folds, namely RNase H, endonuclease, endonuclease VII-colicin E and RusA. The endonuclease fold, whose structural prototypes are the phage lambda exonuclease, the very short patch repair nuclease (Vsr) and type II restriction enzymes, is shown to encompass by far a greater diversity of nucleases than previously suspected. This fold unifies archaeal HJRs, repair nucleases such as RecB and Vsr, restriction enzymes and a variety of predicted nucleases whose specific activities remain to be determined. Within the RNase H fold a new family of predicted HJRs, which is nearly ubiquitous in bacteria, was discovered, in addition to the previously characterized RuvC family. The proteins of this family, typified by Escherichia coli YqgF, are likely to function as an alternative to RuvC in most bacteria, but could be the principal HJRs in low-GC Gram-positive bacteria and AQUIFEX: Endonuclease VII of phage T4 is shown to serve as a structural template for many nucleases, including MCR:A and other type II restriction enzymes. Together with colicin E7, endonuclease VII defines a distinct metal-dependent nuclease fold. As a result of this analysis, the principal HJRs are now known or confidently predicted for all bacteria and archaea whose genomes have been completely sequenced, with many species encoding multiple potential HJRs. Horizontal gene transfer, lineage-specific gene loss and gene family expansion, and non-orthologous gene displacement seem to have been major forces in the evolution of HJRs and related nucleases. A remarkable case of displacement is seen in the Lyme disease spirochete Borrelia burgdorferi, which does not possess any of the typical HJRs, but instead encodes, in its chromosome and each of the linear plasmids, members of the lambda exonuclease family predicted to function as HJRs. The diversity of HJRs and related nucleases in bacteria and archaea contrasts with their near absence in eukaryotes. The few detected eukaryotic representatives of the endonuclease fold and the RNase H fold have probably been acquired from bacteria via horizontal gene transfer. The identity of the principal HJR(s) involved in recombination in eukaryotes remains uncertain; this function could be performed by topoisomerase IB or by a novel, so far undetected, class of enzymes. Likely HJRs and related nucleases were identified in the genomes of numerous bacterial and eukaryotic DNA viruses. Gene flow between viral and cellular genomes has probably played a major role in the evolution of this class of enzymes. This analysis resulted in the prediction of numerous previously unnoticed nucleases, some of which are likely to be new restriction enzymes.
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PMID:SURVEY AND SUMMARY: holliday junction resolvases and related nucleases: identification of new families, phyletic distribution and evolutionary trajectories. 1098 59

NaeI is a type IIe endonuclease that interacts with two DNA recognition sequences to cleave DNA. One DNA sequence serves as a substrate and the other serves to activate cleavage. NaeI is divided into two domains whose structures parallel the two functionalities recognized in NaeI, endonuclease and topoisomerase. In this study, we report evidence for mutations that break interdomain functional communication in a NaeI-DNA complex. Deletion of the initial 124 amino acids of the N-terminal domain of NaeI converted NaeI to a monomer, consistent with self-association being mediated by the Endo domain. Deletions within a small region of the C-terminal DNA binding domain of NaeI (amino acids 182-192) altered the recognition by NaeI of sequences flanking the NaeI recognition sequence. Substituting Ala for Arg182 within this region had no apparent effect on DNA binding but greatly reduced the extent of DNA cleavage even though it is not part of the catalytic Endo domain. Substituting Ala for Ile185 reduced the extent of DNA binding about 1000-fold. Substituting Ala for Lys189 altered flanking sequence recognition. Residues 182-192 are away from the Endo domain responsible for cleavage and also face away from the modeled DNA binding faces of the apoprotein crystal structure. We propose that residues 182-192 are part of a web that mediates the flow of information between the NaeI Endo and Topo domains.
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PMID:Evidence for mutations that break communication between the Endo and Topo domains in NaeI endonuclease/topoisomerase. 1107 9

Type II topoisomerase inhibitors are used to treat both tumors and bacterial infections. These inhibitors stabilize covalent DNA-topoisomerase cleavage complexes that ultimately cause lethal DNA damage. A functional recombinational repair apparatus decreases sensitivity to these drugs, suggesting that topoisomerase-mediated DNA damage is amenable to such repair. Using a bacteriophage T4 model system, we have developed a novel in vivo plasmid-based assay that allows physical analysis of the repair products from one particular topoisomerase cleavage site. We show that the antitumor agent 4'-(9-acridinylamino)methanesulphon-m-anisidide (m-AMSA) stabilizes the T4 type II topoisomerase at the strong topoisomerase cleavage site on the plasmid, thereby stimulating recombinational repair. The resulting m-AMSA-dependent repair products do not form in the absence of functional topoisomerase and appear at lower drug concentrations with a drug-hypersensitive topoisomerase mutant. The appearance of repair products requires that the plasmid contain a T4 origin of replication. Finally, genetic analyses demonstrate that repair product formation is absolutely dependent on genes 32 and 46, largely dependent on genes uvsX and uvsY, and only partly dependent on gene 49. Very similar genetic requirements are observed for repair of endonuclease-generated double-strand breaks, suggesting mechanistic similarity between the two repair pathways.
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PMID:Repair of topoisomerase-mediated DNA damage in bacteriophage T4. 1133 15

Kinetoplast DNA (kDNA), the mitochondrial DNA of the trypanosomatid Crithidia fasciculata, is a unique structure containing 5,000 DNA minicircles topologically linked into a massive network. In vivo, the network is condensed into a disk-shaped structure. Replication of minicircles initiates at unique origins that are bound by universal minicircle sequence (UMS)-binding protein (UMSBP), a sequence-specific DNA-binding protein. This protein, encoded by a nuclear gene, localizes within the cell's single mitochondrion. Using immunofluorescence, we found that UMSBP localizes exclusively to two neighboring sites adjacent to the face of the kDNA disk nearest the cell's flagellum. This site is distinct from the two antipodal positions at the perimeter of the disk that is occupied by DNA polymerase beta, topoisomerase II, and a structure-specific endonuclease. Although we found constant steady-state levels of UMSBP mRNA and protein and a constant rate of UMSBP synthesis throughout the cell cycle, immunofluorescence indicated that UMSBP localization within the kinetoplast is not static. The intramitochondrial localization of UMSBP and other kDNA replication enzymes significantly clarifies our understanding of the process of kDNA replication.
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PMID:Intramitochondrial localization of universal minicircle sequence-binding protein, a trypanosomatid protein that binds kinetoplast minicircle replication origins. 1135 34

Previously we have characterized type IB DNA topoisomerase V (topo V) in the hyperthermophile Methanopyrus kandleri. The enzyme has a powerful topoisomerase activity and is abundant in M. kandleri. Here we report two characterizations of topo V. First, we found that its N-terminal domain has sequence homology with both eukaryotic type IB topoisomerases and the integrase family of tyrosine recombinases. The C-terminal part of the sequence includes 12 repeats, each repeat consisting of two similar but distinct helix-hairpin-helix motifs; the same arrangement is seen in recombination protein RuvA and mammalian DNA polymerase beta. Second, on the basis of sequence homology between topo V and polymerase beta, we predict and demonstrate that topo V possesses apurinic/apyrimidinic (AP) site-processing activities that are important in base excision DNA repair: (i) it incises the phosphodiester backbone at the AP site, and (ii) at the AP endonuclease cleaved AP site, it removes the 5' 2-deoxyribose 5-phosphate moiety so that a single-nucleotide gap with a 3'-hydroxyl and 5'-phosphate can be filled by a DNA polymerase. Topo V is thus the prototype for a new subfamily of type IB topoisomerases and is the first example of a topoisomerase with associated DNA repair activities.
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PMID:A type IB topoisomerase with DNA repair activities. 1135 38

Topoisomerases constitute a family of highly conserved essential enzymes, which exist in all investigated living pro- and eukaryotic cells. They are indispensable for the control of DNA topology. Humans possess 4 types of topoisomerases, i. e. topoisomerase (topo) I, II, III and V. Topo I, a 100-kDa protein, is a member of the type-I enzyme group (type IB). Functionally, it is an ATP-independent DNA single-strand endonuclease and ligase that functions mainly during transcription but also during DNA replication. Topo II belongs to the type-II enzymes and is represented in humans by 2 highly homologous isoforms, alpha (170 kDa) and beta (180 kDa). Contrary to topo I, the 2 topo II isoforms are ATP-dependent double-strand endonucleases and ligases. Topo I and the beta-form of topo II are expressed in a proliferation-independent manner, whereas topo IIalpha is cell-cycle-regulated. Because of the crucial role of topoisomerases for the maintenance and replication of DNA during proliferation, cells become highly vulnerable when these functions are lost. Consequently, a wide range of drugs with cytostatic effects are topo inhibitors. Topo I inhibitors in clinical use belong to the camptothecin family, e. g. topotecan and irinotecan. Topo IIalpha inhibitors are constituents of most chemotherapeutic protocols and form a large heterogeneous group. It includes clinically used compounds such as the podophyllotoxin analogues etoposide and teniposide, the anthracyclines daunorubicin, doxorubicin and idarubicin, the anthracenedione mitoxantrone and amsacrine. Recently, substances with dual specificity that inhibit both topo I and topo IIalpha have been found. The clinical relevance of these new compounds remains to be established. Specific inhibitors of topo IIbeta have not been described yet. The majority of topo inhibitors interfere with the religation step in the normal action of the enzymes, which leads to a stabilisation of the so-called cleavable complex. This results in DNA single-strand breaks in the case of topo I or double-strand breaks in the case of topo II. DNA single-strand breaks due to topo I inhibition are converted into double-strand breaks in the course of DNA replication. Such topo-mediated DNA strand breaks likely induce repair or apoptosis mechanisms via p53 and/or p21(WAF1/Clip1). As a consequence, while topoisomerases are required for proliferation, proliferation is also essential for efficacious topo inhibition. The cell-cycle-dependent expression of topo IIwas also successfully used for prognostic evaluations of survival in patients with cancer. Copyright 2000 S. Karger GmbH, Freiburg
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PMID:Human DNA-Topoisomerases - Diagnostic and Therapeutic Implications for Cancer. 1144 Dec 36

The RecQ DNA helicases, human BLM and yeast Sgs1, form a complex with topoisomerase III (Top3) and are thought to act during DNA replication to restart forks that have paused due to DNA damage or topological stress. We have shown previously that yeast cells lacking SGS1 or TOP3 require MMS4 and MUS81 for viability. Here we show that Mms4 and Mus81 form a heterodimeric structure-specific endonuclease that cleaves branched DNA. Both subunits are required for optimal expression, substrate binding, and nuclease activity. Mms4 and Mus81 are conserved proteins related to the Rad1-Rad10 (XPF/ERCC1) endonuclease required for nucleotide excision repair (NER). However, the Mms4-Mus81 endonuclease is 25 times more active on branched duplex DNA and replication fork substrates than simple Y-forms, the preferred substrate for the NER complexes. We also present genetic data that indicate a novel role for Mms4-Mus81 in meiotic recombination. Our results suggest that stalled replication forks are substrates for Mms4-Mus81 cleavage-particularly in the absence of Sgs1 or BLM. Repair of this double-strand break (DSB) by homologous recombination may be responsible for the elevated levels of sister chromatid exchange (SCE) found in BLM(-/-) cells.
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PMID:Functional overlap between Sgs1-Top3 and the Mms4-Mus81 endonuclease. 1164 Dec 78

Drosophila topoisomerase (topo) IIIbeta is a member of the type IA family of DNA topoisomerases, which generates a single-stranded break to form a covalent complex with the 5'-end of DNA. We show here that a purified preparation of topo IIIbeta is able to convert a hypernegatively supercoiled substrate into primarily nicked, but also linear, DNA at enzyme/DNA molar ratios of 5:1 or greater. Although the optimal temperature for the relaxation activity is between 37 and 45 degrees C, maximal cleavage occurs between 23 and 30 degrees C, a temperature range that is more physiologically relevant for fruit flies. The cleavage products require protease treatment to enter the gel, they are stable over time, they are reversible, and they are not observed with a Y332F active site mutant, which further supports the idea that topo IIIbeta possesses an endonucleolytic cleavage activity. This cleavage activity appears to be specific for highly unwound, or single strand-containing substrates. Southern blot analysis of the cleavage products demonstrates that the topo IIIbeta cleavage activity is concentrated primarily in highly A/T-rich regions. These results suggest that topo IIIbeta may function as a reversible endonuclease in vivo by recognizing and cleaving/rejoining DNA structures with single-stranded character.
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PMID:Generation of double-stranded breaks in hypernegatively supercoiled DNA by Drosophila topoisomerase IIIbeta, a type IA enzyme. 1202 76

The topoisomerase (topo) III enzymes are found in organisms ranging from bacteria to humans, yet the precise cellular function of these enzymes remains to be determined. We previously found that Drosophila topo IIIbeta can relax plasmid DNA only if the DNA is first hypernegatively supercoiled. To investigate the possibility that topo IIIbeta requires a single-stranded region for its relaxation activity, we formed R-loops and D-loops in plasmids. In addition to containing a single-stranded region, these R-loops and D-loops have the advantage of being covalently closed and supercoiled, thus allowing us to assay for supercoil relaxation. We found that topo IIIbeta preferentially cleaves, rather than relaxes, these substrates. The cleavage of the R-loops and D-loops, which is primarily in the form of nicking, occurs to a greater extent at a temperature that is lower than the optimal temperature for relaxation of hypernegatively supercoiled plasmid. In addition, the cleavage can be readily reversed by high salt or high temperature, and the products fail to enter the gel in the absence of proteinase K treatment and are not observed with an active-site Y332F mutant of topo IIIbeta, indicating that the cleavage is mediated by a topoisomerase. We mapped the cleavage to the unpaired strand within the loop region and found that the cleavage occurs along the length of the unpaired strand. These studies suggest that the topo III enzyme behaves as a structure-specific endonuclease in vivo, providing a reversible DNA cleavage activity that is specific for unpaired regions in the DNA.
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PMID:Preferential cleavage of plasmid-based R-loops and D-loops by Drosophila topoisomerase IIIbeta. 1204 41

NaeI endonuclease contains a 10-amino acid region with sequence similarity to the active site KXDG motif of DNA ligase except for leucine (Leu-43) in NaeI ((43)LXDG(46)). Changing Leu-43 to lysine abolishes the NaeI endonuclease activity and replaces it with topoisomerase and recombinase activities. Here we report the results of substituting Leu-43 with alanine, arginine, asparagine, glutamate, and histidine. Quantitating specific activities and DNA binding values for the mutant proteins determined the range of amino acids at position 43 that alter NaeI mechanism. Substituting alanine, asparagine, glutamate, and histidine for Leu-43 maintained endonuclease activity, but at a lower level. On the other hand, substituting positively charged arginine, like lysine at position 43, converted NaeI to a topoisomerase with no observable double-strand cleavage activity. The specific activities of NaeI-43K and NaeI-43R and their relative sensitivities to salt, the topoisomerase-inhibiting drug N-[4-(9-acridinylamino)-3-methoxyphenyl]methane-sulfonamide (amsacrine) and single-stranded DNA showed that the two activities are similar. The effect of placing a positive charge at position 43 on NaeI structure was determined by measuring (for NaeI and NaeI-43K) relative susceptibilities to proteolysis, UV, circular dichroism spectra, and temperature melting transitions. The results provide evidence that a positive charge at position 43 induces dramatic changes in NaeI structure that affect both the Endo and Topo domains of NaeI. The identification of four putative DNA ligase motifs in NaeI leads us to speculate that structural changes that superimpose these motifs on the ligase structure may account for the changes in activity.
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PMID:Amino acid substitutions at position 43 of NaeI endonuclease. Evidence for changes in NaeI structure. 1251 52


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