DNA-topoisomerase

Onishi et al. (1993) developed an assay that monitors the relaxation of super-coiled plasmid DNA induced by DNA topoisomerase. Supercoiling of DNA is important in the formation of chromatin and in determining functions of DNA, including replication, transcription, recombination, and repair. 

Relaxed and supercoiled DNA samples from plasmid pBR329 were separated by ion-exchange chromatography on a TOSO DEAE-NPR column (4.6 mm x 35 mm) or TOSO DEAE-5PW column (7.5 mm X 75 mm). These columns were equilibrated with 20 mM Tris-HCl buffer (pH 7.5) containing 0.5 M NaCl, and the column was eluted with a linear gradient from 0.5 to 0.65 M NaCl at a flow rate of 1 mL/min. Absorbance was monitored at 260 nm. 

The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.5), 120 mM KC1,10 mM MgCfe, 0.5 mM EDTA, 0.5 mM dithiothreitol, 30 /xg/mL bovine serum albumin, and 0.2 fig of pBR329 DNA. After incubation at 37°C for 30 minutes, the reaction was terminated by the addition of Sarkosyl in a final concentration of 1%. The reaction mixture was further incubated at 37°C for 30 minutes after proteinase K had been added to give a concentration of 50 /xg/mL. Assays were linear with up to 0.4 fig of topoisomerase I, and with time until about 95% of the substrate had been converted into relaxed DNA. 

Topoisomerase I was partially purified from the nuclei of HeLa S3 cells by a procedure that involved lysing the nuclei and precipitating nucleic acid with polyethylene glycol. 

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Figure 36-18. Comparison of two types of nick-sealing reactions on DNA. The series of reactions at left is catalyzed by DNA topoisomerase I, that at right by DNA ligase P = phosphate, R = ribose, A = ademine. (Slightly modified and reproduced, with permission, from Lehninger AL Biochemistry, 2nd ed. Worth, 1975.)...

MARKOVITS J, LINASSIER C, FOSSE P, COUPRIE J, PIERRE J, JACQUEMIN-SABLON A, SAUCIER J M, LE PECQ J B, LARSEN A K (1989) Inhibitory effects of the tyrosine kinase inhibitor genistein on mammalian DNA topoisomerase II. Cancer Res. 49 5111-17. 

Deoxyribonucleic acid (DNA) topoisomerases (topos) are ubiquitous enzymes that can manipulate DNA by changing the number of topological links between two strands of the same or different DNA molecules [1]. These enzymes are involved in many cellular processes, such as replication, recom-... 

Peczyfiska-Czoch W et al. (1996) Microbial transformation of azacarbazoles X re-gioselective hydroxylation of 5,ll-dimethyl-5ff-indolo[2,3-l)]quinoline, a novel DNA topoisomerase 11 inhibitor, hy Rhizopus arrhizus. Biotechnol Lett 18(2) 123-128... 

Scheme 5 Structure of several coralyne analogues tested for inhibition of DNA topoisomerase I...

This royal-blue-colored drug is an anthracenedione that inhibits DNA topoisomerase II. The pharmacokinetics of mitoxantrone may best be described by a three-compartment model, with an a half-life of 3 to 10 minutes, a 3 half life of 0.3 to 3 hours, and a median terminal half-life of 12 days. Biliary elimination appears to be the primary route of elimination, with less than 10% of the drug eliminated by the kidney.23 Mitoxantrone has shown clinical activity in the treatment of acute leukemias, breast and prostate cancer, and non-Hodgkin s lymphomas. Myelosuppression, mucositis, nausea and vomiting, and cardiac toxicity are side effects of this drug. The total cumulative dose limit is 160 mg/m2 for patients who have not received prior anthracycline or mediastinal radiation. Patients who have received prior doxorubicin or daunorubicin therapy should not receive a cumulative dose greater than 120 mg/m2 of mitoxantrone. Patients should be counseled that their urine will turn a blue-green color. 

Camptosar ) the covalently bound DNA topoisomerase enzyme and interferes with the DNA breakage-resealing process. Early and late diarrhea Additional toxicities Neutropenia, nausea, and vomiting... 

Irinotecan (Camptosar ) is a topoisomerase I inhibitor that forms a complex with the covalently bound DNA topoisomerase... 

Topoisomerase An enzyme that uncoils DNA during replication by cutting the DNA. Topoisomerase inhibitors prevent DNA from sealing the cut, which causes DNA strand breakage. 

As discussed above, alternative recombinant DNA techniques are necessary to efficiently generate genome-scale clone sets. One alternative exploits the ability of the Vaccinia virus DNA topoisomerase I to both cleave and rejoin DNA strands with high sequence specificity (Shuman, 1992a Shuman, 1992b). In the reaction, the enzyme recognizes the sequence 5 -CCCTT and cleaves at the final T whereby a covalent adduct is formed between the 3 phosphate of the cleaved strand and a tyrosine residue in the enzyme (Fig. 4.1). The covalent complex can combine with a heterologous acceptor DNA that has a 5 hydroxyl tail complementary to the sequence on the covalent adduct to create a recombinant molecule (Shuman, 1994). 

Shuman, S. (1994). Novel approach to molecular cloning and polynucleotide synthesis using vaccinai DNA topoisomerase. J. Biol. Chem. 269, 32678-32684. 

Dinardo S, Voelkel K, Stemglanz R 1984 DNA topoisomerase II mutant of Saccharomyces cerevisiae. topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication. Proc Natl Acad Sci USA 81 2616-2620... 

Downes CS, Mullinger AM, Johnson RT 1991 Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis. Proc Nad Acad Sci USA 88 8895-8899... 

An other example of Salvia quinone is salvicine, a structurally modified diterpenoid quinone derived from Salvia prionitis, which is cytotoxic against multidrug-resistant cancer cell lines of topoisomerase II inhibition by trapping the DNA-topoisomerase II complex (49). 

Tosa H, Iinuma M, Asai F, et al. Anthraquinones from Neonauclea calycina and their inhibitory activity against DNA topoisomerase II. Biol Pharm Bull 1998 21 641-642. 

Wada S, Reiko T, Akira I, Shunyo M. In vitro inhibitory effects of DNA topoisomerase II by femane-type triterpenoids isolated from a Euphorbia genus. Bioorg Med Chem Lett 1998 8 2829-2832. 

Wada S, Iida A, Tanaka R. Screening of triterpenoids isolated from Phyllanthus flexuo-sms for DNA topoisomerase inhibitory activity. J Nat Prod 2001 64 1545-1547. 

Meng LH, Zhang JS, Ding J. Salvicine, a novel DNA topoisomerase II inhibitor, exerting its effects by trapping enzyme-DNA cleavage complexes. Biochem Pharmacol 2001 62 733-741. 

Ataxia telangiectasia mutated (ATM), poly(ADP ribose) polymerase (PARP), DNA-dependent protein kinase, DNA replication factor C, DNA topoisomerase I, DNA fragmentation factor (DFF)45, inhibitor of caspase-activated DNAse (ICAD), lamins A, Bl, and C TRAF-1, Rafl, Ras, GAP, GDP dissociation inhibitor of Rho family GTPases, phospholipase A2, Statl... 

Schultz P, Olland S, Oudet P, Hancock R. Structure and conformational changes of DNA topoisomerase II visualized by electron microscopy. Proc Natl Acad Sci USA 1996 93 5936-5940. 

Furuta T, Takemura H, Liao ZY, Aune GJ, Redon C, Sedelnikova OA, et al. Phosphorylation of histone H2AX and activation of Mrell, Rad50, and Nbsl in response to replication-dependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. J. Biol. Chem. 2003 May 30 278(22) 20303-12. 

Leppard JB, Champoux JJ. Human DNA topoisomerase I relaxation, roles, and damage control. Chromosoma. 2005 Jul 114(2) 75-85. 

Huang X, Okafuji M, Traganos F, Luther E, Holden E, Darzynkiewicz Z. Assessment of histone H2AX phosphorylation induced by DNA topoisomerase I and II inhibitors topotecan and mitoxantrone and by the DNA cross-linking agent cisplatin. Cytometry A. 2004 Apr 58(2) 99-110. 

Adachi Y, Kas E, Laemmli UK (1989) Preferential, cooperative binding of DNA topoisomerase 11 to scaffold-associated regions. Embo J 8 3997 006... 

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