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Dna Topoisomerase

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Last Updated: 02 July 2021

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General | Latest Info

Deoxyribonucleic acid holds all genetic information of cell. It is an instruction manual, providing cells with information on how to grow, function, reproduce and more. It is written in a four - letter alphabet or code. Each letter is known DNA base. Individual cells contain a staggering six billion DNA base pairs, linearly arranged one after another. The total length of DNA within each nucleus is two metres, while the total length of DNA within roughly 37. 2 trillion cells in the human body is 60 billion kilometres. The distance between earth and the moon is merely 384 400 kilometres. How is this possible? Dna double helix has multiple layers of packaging, to fit within a 5 - 10 micrometre - size nucleus. This Three - dimensional folding of DNA is important for how proteins, ions, and other molecules can access and interact with DNA during vital cell processes. Such processes include copying of DNA when cells divide, coding of DNA into RNA as prelude to protein synthesis, and repair of DNA when damage bases are detect. Relax, topoisomerase is here DNA in a cell can over - twist or under - twist, ending up in what we call a supercoiled state where it twists around itself to relieve torsional stress exerted on double helix. Dna can be either negatively supercoiled or positively supercoiled. Extreme compaction of DNA in cells exacerbates issues related to DNA supercoiling. It OK to take a break. Topoisomerases cut DNA to release tension created by twists and turns in double helix. Typically, DNA is negatively supercoiled. This makes it easier to pull two strands of DNA apart. As a result, proteins and other factors can more easily access DNA when needed for important cell processes. However, during events such as replication and transcription, double helix become over - twist, which makes it more difficult to open DNA. This tension inhibits these processes. In addition, during recombination and replication, knots and tangles are introduced into double - helix. Knotting and tangling prevent progression of cell functions. To avoid detrimental consequences, torsional stress as well as knots and tangles in DNA must be manage. This is accomplished by a group of enzymes called Topoisomerases that act by transiently breaking and re - joining DNA. Everything around you is numbers. Precise mathematical expressions are used to describe supercoiling by comparing different coil states to relaxed state of DNA. Decades of scientific research have taught us that topoisomerases distinguish between relaxed and supercoiled DNA and can see and cut specific DNA sequences. For these abilities, Topoisomerases have been referred to as molecular mathematicians of Cell. However, many fundamental questions remain related to the precision and speed of their actions. Dr Neil Osheroff, professor of Biochemistry and Medicine at Vanderbilt University School of Medicine, has dedicated many years to understanding how topoisomerase enzymes work.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Figure Locations

Experimental approaches and validation. Schematic of experimental approaches to mapping TopoI functional sites. Imipramine treat and D108A mutant mediate Protein - DNA covalent adducts were immune - precipated using anti - TopoI antibodies and sequence. Overall occupancy of TopoI is determined by formaldehyde crosslinking of TopoI and DNA. Topoi linked to DNA was immune - precipitate using anti - TopoI antibodies. Dot - blots showing Protein - DNA covalent complexes isolated from MsTopoI D108A overexpressing M. Smegmatis cells and imipramine treat M. Smegmatis cells probe with TopoI antibodies. Qpcr showing increased recA expression in MsTopoI D108A overexpressing M. Smegmatis cells and imipramine treating M. Smegmatis cells. Data represent is mean SD from three independent experiments. Ut = untreated, IT = imipramine treat. Analyses of TopoI binding and cleavage sites distribution in genome. Circos plot for the whole genome of TopoI functional sites. Innermost red circle represents basal TopoI cleavage, green circle depicts D108A induced cleavage and the blue circle is the drug imipramine induce cleavage. The grey circle depicts TopoI occupancy by formaldehyde crosslinked ChIP and the outermost circle represents the genomic coordinates of M. Smegmatis. Ucsc genome browser view of TopoI binding and cleavage peaks across M. Smegmatis genome. The Ucsc browser has higher magnification view of different genomic coordinates. D108a, IT and TopoI FC are mark. Additional drugs induce IT cleavage peaks are seen compared to D108A induced cleavage. In all three genomic coordinates chosen, basal cleavage AT same location as D108A. The right panel shows in vitro DNA cleavage by MsTopoI. Oligonucleotides designed from one of peaks of genomic coordinates 6200 - 6300 4900 - 5100 and 3700 - 3900 kb respectively were analysis for cleavage as described in Materials and Methods. Resultant products were analyse on 8M urea 12% polyacrylamide gel. Arrow indicates cleavage product. Protection of cleavage sites by NAPs. Ucsc browser view in higher magnification for 3905 - 4050 and 5050 - 5200 kb genomic coordinates respectively. Cleavage assay with DNA having TopoI recognition motif but cleavage not seen in vivo. Oligonucleotides from DNA binding peaks were treated with increasing concentration of MsTopoI. Resultant products were analyse on 8M urea 12% polyacrylamide gel. Protection assay with NAPs. 32mer oligonucleotides were incubated with increasing concentration of HU or Lsr2 and cleavage assays were carried out with TopoI. Resultant products were analyse on 8 M urea and 12% polyacrylamide gel. Transcription modulates TopoI activity. The M. Smegmatis genome is divided into windows of 6kb around TSS. Reads falling in each overlap window were normalized to get read count frequency for TopoI binding and RNAP binding. Similarly, normalized read count frequency, TopoI activity and elongating RNAP were plot. Genomic DNA primer extension showing attenuation of in vivo TopoI mediate DNA cleavage by Rifampicin. Genomic DNA was isolated from MsTopoI D108A, MsTopoI D108A treated with Rifampicin before induction of MsTopoI D108A and WT M. Smegmatis, imipramine treat cells and Rifampicin + imipramine treat cells. Primer extension was performed with specific forward primers for 4900 - 5100 kb and 6200 - 6300 kb respectively with Taq DNA polymerase. The resulting products were analyse on 8M urea 12% polyacrylamide gel. Arrow indicates cleavage product. Topoi catalyse catenation and decatenation.

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By Jennifer McDowall

Dna in the nucleus of the cell contains all the information it requires to carry out life processes: growth, development, maintenance, reproduction and protection. With all this information, it is no wonder that the length of cell DNA is far greater than that of the cell compartment that contains it - DNA in a single human diploid cell contains over 7 billion base pairs divided into 46 chromosomes, which would extend over 2 meters in length if stretch end to end. Yet this massive volume of DNA can be condensed to yield highly compact chromosomes through tight packing of DNA: first DNA is wound around protein nucleosomes like beads on string, then beads are coiled into a helical structure, and finally, the helical structure is itself supercoiled into highly - pack coiled - coil structure. This highly compact structure allows DNA to be safely stored in the nucleus, and to be divided up during cell division without damaging DNA. However, such tightly wound DNA does not permit molecules to gain access to individual genes in order to transcribe copies of them, as required for protein synthesis. To overcome this problem, cells use specialised proteins to unwind DNA in specific regions when it needs access to it, while keeping the rest of DNA molecule tightly wound and out of harm's way. Once DNA is uncoiled, DNA double helix itself needs to be unwound to separate it into two individual strands so information it contains can be access. Proteins that carry out this job are collectively known as DNA Topoisomerases.


The X chromosome sequence

We constructed a map of the X chromosome using predominantly P1 - artificial chromosome and bacterial artificial chromosome clones, which were assembled into contigs using restriction - enzyme fingerprinting and integrated with earlier maps using sequence - tag site content analysis 5. Gaps were closed by targeted screening of clone libraries in bacteria or yeast, and by assessing BAC and fosmid end - sequence data for evidence of spanning clones. Fourteen euchromatic gaps remain intractable, despite using libraries with combined 80 - fold chromosome coverage. Five of these gaps are within 2. 7 megabase pseudoautosomal region at tip of chromosome short arm. This is reminiscent of situation in other human sub - telomeric regions 6, and might reflect cloning difficulties in areas with high content of nucleotides and minisatellite repeats. We selected 1 832 clones from the map for shotgun sequencing and direct finishing using established procedures 7. Finish sequences were estimated to be more than 99. 99% accurate by independent assessment 8. The sequence of X chromosome has been assembled from individual clone sequences and comprises 16 contigs. These extend into telomeric n repeat arrays at the ends of chromosome arms, and include both pseudoautosomal regions. Data were frozen for analyses described below, at which point we had determined 150 396 262 base pairs of sequence. Subsequently, we obtained a further 609 664 bp of sequence. 14 euchromatic gaps are estimated to have a combined size of less than 1 Mb, and sequence therefore cover at least 99. 3% of X chromosome euchromatin. There is also a single heterochromatic gap corresponding to polymorphic 3. 0 Mb array 9 of alpha satellite DNA at centromere. On this basis, we conclude that the X chromosome is approximately 155 Mb in length. Coverage and quality of finish sequence have been assessed using independent data. All markers from deCODE genetic map 10 are found in sequence and concordance of marker orders is excellent with only one discrepancy. Dxs6807 is the most distal Xp marker on the deCODE map, but in sequence this marker is proximal to three others with genetic locations of 9 - 11 cM on the deCODE map. Out of 788 X chromosomal RefSeq 11 messenger RNAs that were assess, 783 were found completely in sequence, and parts of four others were also present. Missing segments of GTPBP6, CRLF2, DHRSX and FGF16 lie within gaps 1 4 5 and 10, respectively, and the GAGE3 gene is in gap 7. Sequence assembly was assessed using fosmid end - sequence pairs that match X chromosome sequence. Orientation and separation of end - pairs of more than 17 000 fosmids were consistent with sequence assembly. In two cases, sequences had been misassembled owing to long and highly similar repeats. There were six instances of large deletions in sequenced clones, which were resolved by determining fosmid sequences through deleted regions. Finally, there were two cases of apparent length variation between reference sequence and DNA use for fosmid library.


Genes

The gene set described above includes non - coding RNA genes only when there is supporting evidence of expression from complementary DNA or express - sequence - tag sources. Using a complementary approach, we analyse X chromosome sequence using the Rfam 15 database of structural RNA alignments, and predict 173 ncRNA genes and / or pseudogenes. These are physically separate from genes described in the preceding section and are not included in total gene count, owing to difficulty in discriminating between genes and pseudogenes for these ncRNA predictions. Using tRNAscan - SE 16, we predict only two transfer RNA genes on the X chromosome, out of several hundred predicted in human genome 7. Thirteen microRNAs from microRNA registry 17 have also been mapped onto sequence. The most prominent of ncRNA genes on the X chromosome is XIST 18, which is critical for XCI. Xist locus spans 32 103 bp in Xq13, and its untranslated transcript coats and transcriptionally silences one X chromosome in cis. Refseq 11 transcript of XIST is RNA of 19 275 bases, which includes the largest exon on chromosome. There is also evidence for shorter XIST transcripts generated by alternative splicing, particularly in 3 regions of gene 19. In mouse, TSIX is antisense to XIST 20, and its transcript is believed to repress accumulation of XIST RNA. There is evidence for transcription antisense to XIST in human 21 22, but we have been unable to annotate human TSIX gene as there are no corresponding express sequences in public databases, and because there is lack of primary sequence conservation between human and mouse regions. In human sequence, two other ncRNA genes are annotated in a 400 kb region distal to XIST, which are orthologues of mouse genes described previously as Jpx and Ftx. In mouse, XIST, Jpx and Ftx are located within smaller area of approximately 200 kb 23.

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MATERIALS AND METHODS

Crystals were tested in - house for diffraction quality using Oxford Xcalibur Nova CCD diffractometer and then transported for high - resolution data collection at Diamond Light Source using ADSC quantum 315 detectors. Data was integrated and reduced using HKL2000. To our surprise, levofloxacin - soak crystals exhibit good quality diffraction and relatively high intensity of diffracted reflections at high angle, exceeding that of one of best native PD 0305970 - containing crystals and all other soaked crystals. Diffraction reached a resolution of 2. 9 with some anisotropic data extending up to 2. 6. The best dataset obtained so far for native PD 0305970 - containing crystal extends up to 3. 1. Crystals soaked using other drugs as well as crystals from which PD 0305970 was back - soak out, diffract to lower resolution and do not show clear and interpretable drug envelope in 2F obs - F calc maps after refinement. By contrast, EDTA and EDTA - Mg crystals exhibit diffraction patterns of relatively high intensity and resolution of data obtained extends up to 3. 3 and 3. 5 respectively. The Highest resolution dataset was used to obtain structure solution and refinement. Structure was solved by molecular replacement in Phaser using as search models our cleavage complex of Topo IV from S. Pneumoniae with moxifloxacin. Refinement was performed in Phenix using secondary structure restraints derived from previously solved complex. Rigid body, simulate annealing, positional and TLS refinement have been perform. Refine Model with Drug molecules omit was used to solve structures of native PD 0305970 - containing crystals as well as EDTA and EDTA - Mg crystals. For all these additional structures, rigid body refinement, simulate annealing, positional and TLS refinement were perform. Drug molecules, magnesium ions and water molecules were located during the last stages of refinement according to missing electron density in - weight 2F obs - F calc and F obs - F calc maps. Wincoot was used for interactive model fitting. Structures were verified using WinCoot and ProCheck,. The Models had good geometry with 82. 5 / 16. 2 / 1. 0 86. 8 / 11. 8 / 1. 1 83. 9 / 13. 8 / 1. 6 and 82. 4 / 15. 4 / 1. 4 percent of residues in favoured / allow / generously allow regions of Ramachandran plot, respectively, and no more than 0. 3 - 0. 7% of residues in disallowed regions. Final Data collection refinement statistics are given in Table S1. Dna conformation was analyse using 3DNA,. Figures were prepared using PyMOL. To our surprise, EDTA - soaked crystals do not lose Mg 2 + ion as it could still be found in electron density coordinating several negatively charged residues such as Glu 433, Asp 506 as well as 5 phosphate groups. Drug had been completely remove, however, DNA was still cloven and covalently attached to active site tyrosines. A similar situation was observed for EDTA - Mg crystals, although in this case DNA was re - seal.


Introduction

Bacterial type II enzymes topoisomerase IV and gyrase have important roles in DNA replication -. Topoisomerase IV, tetramer of two ParC and two ParE subunits, unlinks daughter chromosomes prior to cell division whereas related enzyme gyrase, GyrA 2 GyrB 2 tetramer, supercoils DNA and helps unwind DNA at replication forks. Both enzymes act via double - strand DNA break involving the cleavage complex. Detailed understanding of their reaction pathway has been frustrated by the transient nature of this key intermediate and the absence of relevant drug - free structures. Antibacterial fluoroquinolones stabilize the cleavage complex in an Mg 2 + - dependent fashion, facilitating biochemical and structural work,. Recent studies have also identified quinazolinediones as a new class of topoisomerase IV inhibitors with activity against Streptococcus pneumoniae and other Gram - positive pathogens -. Here we use novel quinazolinedione to generate crystals of Streptococcus pneumoniae topoisomerase IV capture as cleavage complex, and by exploiting slow resealing of complex, we have now obtained first diffracting crystals of sequential catalytically - competent drug - free - DNA cleave and reseal complexes. The structures of these complexes explain unique dione activities and provide the structural basis for reversible DNA strand breakage by type II topoisomerase.


Results and Discussion

Braiding Two DNA Molecules: Experiments and Model. We examined decatenase activity of two different topoisomerases by monitoring in real time increase in extension of molecules as they were unlinked. Molecules extension z to give force F was measured as described in Materials and Methods. Because the elastic behavior of single stretched DNA molecule is well know, we verified that the bead was tethered by two molecules by comparing measure extension z max in abscence of braiding with expect extension for bead tether by two molecules. Molecules were then braid around each other N times by rotating magnets, resulting in a decrease in z. Variation of z with N is nicely described by geometric braiding of two ropes of radius R and maximal extension z max anchor distance 2 E apart as in swing. This model implies that, in contrast with previous single - molecule supercoiling experiments, braid angle is related to molecule extension by cos = z / z max. At any constant force F, extension z shrinks with N, whereas increases until strands are in close contact at N = N C where = C = 45. As we further twist molecules and bring them into closer contact, braiding torque increases until at N = N B, braids buckle and form plectonemic supercoils. This transition is observed when z / z max = z B / z max 0. 64 and is characterized by increase in fluctuations in extension and by discontinuity in slope of z / z max vs. N. That buckling transition was also observed in numerical simulations of braiding of two stretched DNA Molecules. On further twisting, extension z shortened by a constant amount z 42 nm per turn, as expected for plectonemic braids. At each force F, we determine values of intermolecular distance 2 E and DNA's effective radius R that best fit data for cos to Model when < 45. That best - fit values of 2 E vary little over factor 10 variation in F further validates our Model. Furthermore, as predicted by Marko, at large forces braid radius R decreased to F - 3 / 4 due to reduction in entropic confinement. Its value at F = 0. 3 pN is compatible with values measured on free plasmids in similar salt conditions. Excellent agreement between braiding data and the simple geometric model just described allows for detailed understanding of DNA unbraiding by type - II topoisomerases. Eukaryotic Topo Decatenates L - and R - Braids at Same Rate as It relaxes Supercoiled DNA. The Addition of Eukaryotic Topo II in appropriate reaction buffer leads to an increase in extension corresponding to decatenation of L - or R - Braids.

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RESULTS AND DISCUSSION

Validation of purified singly - link catenanes as substrates for topoisomerase - catalyze decatenation reactions was carried out by analyzing decatenation activity of enzymes using new substrate in direct comparison with kDNA. Substrates were tested using two DNA topoisomerases, possessing differing decatenation efficiencies. Topo II is a type II topoisomerase that controls and manipulates the topological state of DNA, principally during processes of chromosome condensation, chromatid segregation, transcription and translation, by initiating transient double - strand DNA breaks. Topo II has a variety of activities, including the ability to relax both positively and negatively supercoiled DNA and interconvert catenate and decatenated, or knot and unknotted, DNA forms. Direct comparison of singly - link catenanes and kDNA as substrates for topo II - mediate decatenation reveals that the former substrate produces more discernible and sensitive decatenation assay when compared with kDNA. Singly - link catenanes can be clearly visualized on agarose gels, prior to addition of enzyme, AT which point kDNA is trapped in wells of gel. Addition of topo II leads to decatenation of singly - linked catenanes in a single step to release two separate circular DNA molecules of visibly distinguishable sizes: 2. 6 and 2. 3 kbp. In contrast, reaction of topo II with kDNA leads to appearance of several DNA molecules of varying sizes on agarose Gel; These are likely to be partially decatenated products. Impressively, use of singly linked DNA catenanes has significantly improved sensitivity of DNA decatenation assay by unambiguously demonstrating full decatenation with 16 - folds less enzyme than required to fully decatenate equal amount of kDNA. In addition, new substrate described here allows for visual distinction between two different activities of topo II. Under these assay conditions, AT topo II concentrations of 13 - 65 pM, decatenation of singly - link dimers into two independent supercoiled DNA molecules is see. Further, the addition of topo II in excess of 130 pM reveals DNA relaxation activity of the enzyme, converting unlinked supercoiled DNA molecules into relaxed forms with relatively lower mobility in agarose Gel. This illustrates how bis - cat assay can be used to simultaneously monitor decatenation and relaxation, showing, in this CASE, that topo II is preferential decatenase; such distinction is not apparent using kDNA. E. Coli DNA gyrase, type II topoisomerase, is distinguished by its unique ability to negatively supercoil DNA in ATP - dependent reaction. Enzymes have also been shown to possess decatenation activity, albeit inefficient in comparison with other type II topoisomerases. Singly - link DNA catenanes and kDNA were subject to relatively modest decatenation activity of gyrase to test the effectiveness of each substrate. Evidently, increased sensitivity provided by reaction on singly linked DNA catenanes readily allows detection of gyrase decatenation activity AT lower concentrations of enzyme, resulting in two distinct bands on agarose Gel, representing separate 2. 6 and 2. 3 kbp supercoiled DNA molecules. Singly - link DNA catenane shows promise for both reassessment of previously identified topoisomerases and characterization of novel decatenation activities.

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CONCLUSION

A partial DNA duplex containing central cleavage site for hTopoII and flanking FRET probes was prepare. Duplex has biotinylated single - strand overhang to avoid any potential steric hindrance caused by surface immobilization. After immobilizing DNA molecules on a polymer - coated quartz surface, hTopoII was delivered into the detection chamber, and fluorescence signals of single DNA molecules were monitored using a total - internal - reflection fluorescence microscope. In the absence of enzyme, fluorescence intensities show single - state behavior with small fluctuations limited by shot - noise. Upon addition of hTopoII, however, large intensity jumps with appreciable dwell times were observe. The frequency of intensity jumps increases linearly with enzyme concentration, while lifetime of high intensity state does not show any appreciable change over the examined enzyme concentration range. Thus, we infer that intensity jumps correspond to binding events of single enzymes. Association rate constant obtained from enzyme titration experiments in Fig. 1 D shows that the association step is diffusion limited. Thus, hindrance of DNA - enzyme interactions by dye - labeling or surface immobilization is negligible. This conclusion is further supported by the fact that similar dissociation constants were obtained in single - molecule and bulk studies. As expected from diffusion - limited binding event, association rate was similar at varying salt concentrations. The Dissociation rate, however, rapidly decreases at lower salt concentrations, indicating that the dissociation constant is very sensitive to ionic strength. Remarkably, in the above experiments, which were performed in the absence of divalent ions, FRET efficiencies remain constant during repeated association / dissociation events of enzyme. In the presence of 5 mM Mg 2 +, however, situation changes dramatically. Large FRET jumps were observed in some of enzyme - DNA binding events, indicating that a substantial amount of DNA deformation was stochastically induced by hTopoII. Similar FRET jumps were observed in the presence of Ca 2 +. Fret jump, however, was not observed when noncleavable DNA sequence was used as substrate, but became more frequent and stable when nick was introduced in one of two scissile bonds of cleavage sequence. Introduction of nick in non - clv, however, does not induce any FRET jump during enzyme binding events. Combining the above observation with knowledge that nick in one strand greatly increases cleavage efficiency of the opposite strand, we conclude that DNA deformation induced by hTopoII occurs in sequence - specific fashion with strong correlation between cleavage efficiency and population of deformed state. Sharp bending of G - segment DNA is supported by a number of experiments including recent high - resolution X - ray structures, atomic force microscopy, and FRET. With this context in mind, FRET jumps are specifically observed only in cleavable sequences in Fig. 2 are interpreted as reflecting sharp bending of G - segment DNA induced by hTopoII.


Introduction

Type IIA topoisomerases are essential DNA - manipulating enzymes ubiquitous in eukaryotes and bacteria 1 2 3. These enzymes exploit protein conformational changes driven by ATP binding and hydrolysis to direct crossing of two DNA duplexes, leading to topological inversion of DNA crossovers 2 4. This unique duplex DNA passage activity allows resolution of intra - and intermolecular DNA entanglements that arise from cellular DNA transactions, including replication, transcription, chromosome segregation, and recombination 1 4 5. Top2 - mediate DNA topological transformation requires temporary creation of DNA double - strand break on one DNA segment, so that another duplex strand can be transported through 2 3 5. To produce this essential DSB, two symmetric Top2 first associated with the G - segment through positively charged groove formed by WHD, tower, and TOPRIM domains 6 7 8. A pair of catalytic tyrosines from WHD domains then initiate transesterification reaction by attacking two 4 - bp stagger phosphodiester bonds on opposite DNA strands, giving rise to the Top2 cleavage complex which harbors a so - called DNA - gate featuring cleave G - segment with enzyme covalently attached to 5 - ends via phosphotyrosyl linkage 9 10 11 12. Next, two N - terminal ATPase domains, which together function as ATP - operated gate, undergo nucleotide - dependent closure to capture and drive T - segment through DNA - gate 13 14 15. Enzyme is reset for its next catalytic cycle by subsequent steps 3 4 6, which include release of T - segment through C - gate form by helical domain append to coiled - coil linker, religation of cleave G - segment by reversal of transesterification reaction, and re - opening of N - gate dimer interface upon ATP hydrolysis. Because directional transport of the T - segment is achieved via coordinated opening and closure of N - gate, DNA - gate, and C - gate, full understanding of the catalytic mechanism of Top2 requires structural characterization of these three gates in distinct conformational states. Crystallographic studies have revealed structural details of N - gate in its open 16 and nucleotide - bound, close 17 18 19 forms. C - gate has also been resolved in both open 8 20 and close 11 12 conformation. In contrast, whereas multiple crystal structures are available for closed DNA - gate 8 11 12 21 22 23, opening of DNA - gate has never been directly visualize. Consequently, outstanding issues regarding operation of DNA - gates remain poorly define, including architectural and surface features of T - segment - conducting path, tertiary and quaternary structural changes associated with gate - opening, and structural changes of enzyme - link DNA cohesive ends upon their detachment from one another. Thus, step - by - step description of how the T - segment is transported through DNA - gate is not yet available. In addition, various clinically active anticancer drugs and antibacterials act by targeting Top2 DNA - gate to produce cytotoxic DNA lesions 3 24 25. Obtaining a more complete picture of the conformational landscape of DNA - gate will contribute to development of new Top2 - targeting agents. We report herein high - resolution view of the opening of Top2 DNA - gate, which directly mediates resolution of topological strand crossings.


Topoisomerases

Dna topoisomerases are able to solve topological problems resulting from replication, transcription, recombination, and reorganization of chromatin. Further on, topoisomerases change the state of supercoiling of DNA and, therefore, have great impact on gene activity. In order to decrease gene activity, DNA topoisomerases introduce temporary single - strand breaks or double - strand breaks in the phosphate backbone of DNA. The Mechanism of topoisomerase action includes transient formation of ester bond between tyrosine residue of enzyme and DNA molecule. Later on, breaks are closed by reformation of original phosphodiester - bond and enzyme release from DNA.


Topoisomerase Inhibitors

Topoisomerase enzymes are important for proper activity of RNA and DNA polymerase. Polymerases separate DNA strands to transcribe base codes. As polymerase separates segments of DNA, remaining portions of strands become more densely coil. Topoisomerase enzymes cleave hypercoiled segments of DNA, relax DNA strands, and then reattach cleaved ends, thereby allowing transcription to progress. There are two classes of topoisomerase enzymes separated by whether they cleave one strand of DNA or both strands as they relieve the hypercoiled state of native DNA. Inhibitors of topoisomerase are specific to type I or type II. The two most common topoisomerase type I inhibitors are camptothecin derivatives topotecan and irinotecan. Topotecan is used in the treatment of ovarian cancer, small cell lung cancer, cervical and renal cell cancers, as well as leukemias and lymphomas. Irinotecan is used for colorectal cancers. Major adverse effects include immunosuppression and, especially with irinotecan, diarrhea. Diarrhea from irinotecan can be severe, and anesthetists might anticipate hypovolemia, metabolic acidosis, and hypokalemia after recent treatments. Among the most common topoisomerase II inhibitors are epipodophyllotoxins, and the group of antineoplastic antibiotics mentioned earlier. The major risk of type II topoisomerase inhibitors is related to the fact that they also produce oxygen free radicals, as previously described for anthracycline antibiotics, leading to cardiotoxicity.

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Personal Response

The Existence of topoisomerases is necessitated by the structure of the double helix. Each human cell contains 2 M of DNA that is compacted into a nucleus that is 10 M in diameter. Because genetic material is anchored to chromosome scaffold and two strands of double helix are plectonemically coil, accessing the genome is a complex topological challenge. Topological properties of DNA are those that can only be changed when the double helix is break. Two aspects of DNA topology significantly affect nuclear processes. The first deals with topological relationships between two strands of double helix. In all living systems, from bacteria to humans, DNA is globally underwound by 6%. This is important because duplex DNA is merely a storage form for genetic information. In order to replicate or express this information, two strands of DNA must be separate. Since global underwinding of genome imparts increased single - strand character to double helix, negative supercoiling greatly facilitates strand separation. While negative supercoiling promotes many nucleic acid processes, DNA overwinding inhibits them. Linear movement of tracking enzymes, such as helicases and polymerases, compresses turns of double helix into shorter region. Consequently, double helix become increasingly overwound ahead of tracking systems. Positive supercoiling that results makes it more difficult to open two strands of double helix and ultimately blocks essential nucleic acid processes. The second aspect of DNA topology deals with relationships between separate DNA segments. Intramolecular knots are generated during recombination, and intermolecular tangles are produced during replication. Dna knots block the essential nucleic acid process because they make it impossible to separate two strands of double helix. Moreover, tangled DNA molecules cannot be segregated during mitosis or meiosis. Consequently, DNA knots and tangles can be lethal to cells if they are not resolve.


TOPOISOMERASE II

Proliferating cells cannot exist without type II topoisomerases. However, since these enzymes generate obligatory double - strand DNA breaks as part of their reaction mechanism, they are intrinsically dangerous proteins. Thus, topoisomerase II assumes the Dr. Jekyll / Mr. Hyde character; while essential to cell viability, enzyme also has the capacity to fragment genome. Because of this dual persona, levels of cleavage complexes are maintained in critical balance. When levels drop below threshold concentrations, daughter chromosomes remain entangled following replication. As a result, chromosomes cannot segregate properly during mitosis and cells die as a result of catastrophic mitotic failure. When levels of cleavage complexes rise too high, cells also die, but for different reasons. Accumulate topoisomerase II - DNA cleavage intermediates are converted to permanent strand breaks when replication forks, transcription complexes or DNA tracking enzymes such as helicases attempt to traverse covalently bind protein roadblock in genetic material. The resulting collision disrupts cleavage complexes and ultimately converts transient topoisomerase II - associate DNA breaks to permanent double - strand breaks that are no longer tethered by proteinaceous bridges. Resulting damage and induction of recombination / repair pathways can trigger mutations, chromosomal translocations and other aberrations. When these permanent DNA breaks are present in sufficient numbers, they can overwhelm cells and initiate death pathways in eukaryotes.

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V Perspectives

Many spectators of the linking - ring trick have been fascinated by magician's seemingly impossible feat of linking solid rings into chain, or separating chain into individual rings. In reality, magicians create the illusion of interconversion by sleight of hand: some rings are permanently linked and others are permanently separate - one exception being a key ring with a hidden opening, through which other rings can be inserted or remove. Dna Topoisomerases are true magicians of the DNA world. In their presence, DNA strands or double helices can pass through each other as if all physical boundaries had disappear: intertwined parental strands of replicating DNA rings can come apart, interlocked double - strand DNA rings can become unlinked and knots can be introduced or removed from DNA rings. In contrast to the hocus - pocus of magician, however, DNA Topoisomerases accomplish their feats by simple and elegant chemistry of transesterification. In strand - breakage reaction by DNA topoisomerase, tyrosyl oxygen of enzyme attacks DNA phosphorus, forming covalent phosphotyrosine link and breaking DNA phosphodiester bond at same time. Rejoining of DNA strand occurs by second transesterification, which is basically reverse of first - oxygen of DNA hydroxyl group that is generated in first reaction attacks phosphorus of phosphotyrosine link, breaking the covalent bond between protein and DNA, and re - forming the DNA backbone bond. These reactions create transient enzyme - mediate gates in DNA for passage of another DNA strand or double helix. Dna Topoisomerases fall into two categories - type I and type II. For type I enzymes, DNA strands are transiently broken one at time; for type II enzymes, by contrast, pair of strands in the DNA double helix are transiently broken in concert by dimeric enzyme molecule. Two types can be further divided into four subfamilies: IA, IB, IIA and IIB. Members of the same subfamily are structurally and mechanistically similar, whereas those of different subfamilies are distinct. The purpose of this review is to provide a perspective of the cellular roles of these remarkable enzymes from the view of their basic reaction characteristics - more comprehensive coverage of literature can be found in several recent reviews. To provide a necessary backdrop, some unique aspects of reactions that are catalyse by different subfamilies of DNA topoisomerases are summarized first. For clarity, reactions that are catalyse by these enzymes are often described for DNA rings. However, similar reactions occur in linear chromosomes owing to their organization into intracellular structures that contain multiple loops, or because their ends are immobile. Type IA. In relaxation of underwound or NEGATIVELY SUPERCOILED DNA by type IA enzyme, short stretch of double - strand DNA is first unpaired by.

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* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

I Introduction

Topoisomerases have evolved to solve topological problems arising from various genetic processes on double - helical DNA. While DNA topoisomerases are essential for DNA replication due to topological intertwining of parental DNA, roles of DNA topoisomerases in transcription are much less clear. However, increasing evidence indicates that topoisomerases may be involved in modulating supercoiling generated during transcription elongation. Because supercoiling of DNA is an important property of DNA and affects almost every aspect of DNA function, roles of topoisomerases in transcription are expectedly complex. It will not be possible to completely understand DNA function without having complete knowledge of the roles of topoisomerases. Additional research efforts are needed in this area.


DNA Topoisomerase

There are a number of different types of topoisomerases, each specialising in different aspects of DNA manipulation. During transcription and DNA replication, DNA needs to be unwound in order for transcription / replication machinery to gain access to DNA so it can be copied or replicate, respectively. Topoisomerase I can make single - strand breaks to allow these processes to proceed. During transcription and DNA replication, DNA helix can become either over - wound or under - wound. For instance, during DNA replication, progress of replication fork generates positive supercoils ahead of replication machinery and negative supercoils behind it. Such tensional problems also exist when transcribing DNA to make RNA copy for protein synthesis. During these processes, DNA can be supercoiled to such an extent that if left unchecked it could impede progress of protein machinery involve. This is prevented by topoisomerase I, which makes single - strand nicks to relax the helix. Before chromosomes separate from one another during cell division, they were able to exchange genetic information through a process known as recombination, where physical pieces of DNA on one chromosome can be swapped for DNA on matching sister chromosome in order to shuffle genetic information. Topoisomerase III can introduce single - strand breaks that are required for DNA to be exchanged by adjacent chromosomes. During the cell cycle, chromosomes must be alternatively condensed and decondensed at specific stages. Topoisomerase II acts as a molecular motor, using energy gained from ATP hydrolysis to introduce tight supercoils into the DNA helix in order to condense chromosome.S Because this process must be highly regulate, topoisomerase II can form molecular complexes with important cell cycle regulators to ensure that chromosome condensation occurs at correct time in cell cycle. During cell division, once chromosomes have been replicate, they must separate and travel to opposite ends of the cell to become part of two separate daughter cells. Topoisomerases IV acts to disentangle replicated daughter strands by making double - strand breaks that allow one duplex to pass through other.

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* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

B DNA Topoisomerase Inhibitors

Dna topoisomerases unravel twists in DNA that occur as a result of DNA transcription and replication. Dna topoisomerases I and II present in cells act through scission of DNA backbone on one or two strands, respectively, followed by relief of torsional stress and then relegation of broken DNA backbone. These enzymes are present in large complexes in the cell nucleus and control and carry out transcription and replication; They are also essential to maintain chromatin organization and cell survival. Inhibition of DNA topoisomerases by small molecules is an effective method for causing DNA damage due to formation of irreversible covalent cross - links between topoisomerase and DNA, stalling its replication and thereby leading to cell death. Among inhibitors of DNA topoisomerase, which are widely used for cancer treatment, are plant alkaloids and antibiotic compounds listed in the following paragraphs.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

Sources

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions.

* Please keep in mind that all text is machine-generated, we do not bear any responsibility, and you should always get advice from professionals before taking any actions

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