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. 1998 Apr 1;26(7):1668–1674. doi: 10.1093/nar/26.7.1668

Sequence specific interaction of Mycobacterium smegmatis topoisomerase I with duplex DNA.

T Bhaduri 1, D Sikder 1, V Nagaraja 1
PMCID: PMC147473  PMID: 9512537

Abstract

We have identified strong topoisomerase sites (STS) for Mycobacteruim smegmatis topoisomerase I in double-stranded DNA context using electrophoretic mobility shift assay of enzyme-DNA covalent complexes. Mg2+, an essential component for DNA relaxation activity of the enzyme, is not required for binding to DNA. The enzyme makes single-stranded nicks, with transient covalent interaction at the 5'-end of the broken DNA strand, a characteristic akin to prokaryotic topoisomerases. More importantly, the enzyme binds to duplex DNA having a preferred site with high affinity, a property similar to the eukaryotic type I topoisomerases. The preferred cleavage site is mapped on a 65 bp duplex DNA and found to be CG/TCTT. Thus, the enzyme resembles other prokaryotic type I topoisomerases in mechanistics of the reaction, but is similar to eukaryotic enzymes in DNA recognition properties.

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Selected References

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  1. Adams D. E., Shekhtman E. M., Zechiedrich E. L., Schmid M. B., Cozzarelli N. R. The role of topoisomerase IV in partitioning bacterial replicons and the structure of catenated intermediates in DNA replication. Cell. 1992 Oct 16;71(2):277–288. doi: 10.1016/0092-8674(92)90356-h. [DOI] [PubMed] [Google Scholar]
  2. Andersen A. H., Gocke E., Bonven B. J., Nielsen O. F., Westergaard O. Topoisomerase I has a strong binding preference for a conserved hexadecameric sequence in the promoter region of the rRNA gene from Tetrahymena pyriformis. Nucleic Acids Res. 1985 Mar 11;13(5):1543–1557. doi: 10.1093/nar/13.5.1543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhaduri T., Nagaraja V. DNA topoisomerase I from Mycobacterium smegmatis. Indian J Biochem Biophys. 1994 Aug;31(4):339–343. [PubMed] [Google Scholar]
  4. Bonven B. J., Gocke E., Westergaard O. A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in Tetrahymena R-chromatin. Cell. 1985 Jun;41(2):541–551. doi: 10.1016/s0092-8674(85)80027-1. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Carey J. Gel retardation at low pH resolves trp repressor-DNA complexes for quantitative study. Proc Natl Acad Sci U S A. 1988 Feb;85(4):975–979. doi: 10.1073/pnas.85.4.975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cove M. E., Tingey A. P., Maxwell A. DNA gyrase can cleave short DNA fragments in the presence of quinolone drugs. Nucleic Acids Res. 1997 Jul 15;25(14):2716–2722. doi: 10.1093/nar/25.14.2716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cozzarelli N. R. DNA gyrase and the supercoiling of DNA. Science. 1980 Feb 29;207(4434):953–960. doi: 10.1126/science.6243420. [DOI] [PubMed] [Google Scholar]
  9. Dean F. B., Cozzarelli N. R. Mechanism of strand passage by Escherichia coli topoisomerase I. The role of the required nick in catenation and knotting of duplex DNA. J Biol Chem. 1985 Apr 25;260(8):4984–4994. [PubMed] [Google Scholar]
  10. Der Garabedian P. A., Mirambeau G., Vermeersch J. J. Mg2+, Asp-, and Glu--effects in the processive and distributive DNA relaxation catalyzed by a eukaryotic topoisomerase. Biochemistry. 1991 Oct 15;30(41):9940–9947. doi: 10.1021/bi00105a018. [DOI] [PubMed] [Google Scholar]
  11. DiNardo S., Voelkel K. A., Sternglanz R., Reynolds A. E., Wright A. Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell. 1982 Nov;31(1):43–51. doi: 10.1016/0092-8674(82)90403-2. [DOI] [PubMed] [Google Scholar]
  12. Edwards K. A., Halligan B. D., Davis J. L., Nivera N. L., Liu L. F. Recognition sites of eukaryotic DNA topoisomerase I: DNA nucleotide sequencing analysis of topo I cleavage sites on SV40 DNA. Nucleic Acids Res. 1982 Apr 24;10(8):2565–2576. doi: 10.1093/nar/10.8.2565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fisher L. M., Barot H. A., Cullen M. E. DNA gyrase complex with DNA: determinants for site-specific DNA breakage. EMBO J. 1986 Jun;5(6):1411–1418. doi: 10.1002/j.1460-2075.1986.tb04375.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
  15. Gmünder H., Kuratli K., Keck W. In the presence of subunit A inhibitors DNA gyrase cleaves DNA fragments as short as 20 bp at specific sites. Nucleic Acids Res. 1997 Feb 1;25(3):604–611. doi: 10.1093/nar/25.3.604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Higgins N. P., Cozzarelli N. R. The binding of gyrase to DNA: analysis by retention by nitrocellulose filters. Nucleic Acids Res. 1982 Nov 11;10(21):6833–6847. doi: 10.1093/nar/10.21.6833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jeltsch A., Maschke H., Selent U., Wenz C., Köhler E., Connolly B. A., Thorogood H., Pingoud A. DNA binding specificity of the EcoRV restriction endonuclease is increased by Mg2+ binding to a metal ion binding site distinct from the catalytic center of the enzyme. Biochemistry. 1995 May 9;34(18):6239–6246. doi: 10.1021/bi00018a028. [DOI] [PubMed] [Google Scholar]
  18. Kirkegaard K., Pflugfelder G., Wang J. C. The cleavage of DNA by type-I DNA topoisomerases. Cold Spring Harb Symp Quant Biol. 1984;49:411–419. doi: 10.1101/sqb.1984.049.01.047. [DOI] [PubMed] [Google Scholar]
  19. Kirkegaard K., Wang J. C. Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop. J Mol Biol. 1985 Oct 5;185(3):625–637. doi: 10.1016/0022-2836(85)90075-0. [DOI] [PubMed] [Google Scholar]
  20. Kostrewa D., Winkler F. K. Mg2+ binding to the active site of EcoRV endonuclease: a crystallographic study of complexes with substrate and product DNA at 2 A resolution. Biochemistry. 1995 Jan 17;34(2):683–696. doi: 10.1021/bi00002a036. [DOI] [PubMed] [Google Scholar]
  21. Kovalsky O. I., Kozyavkin S. A., Slesarev A. I. Archaebacterial reverse gyrase cleavage-site specificity is similar to that of eubacterial DNA topoisomerases I. Nucleic Acids Res. 1990 May 11;18(9):2801–2805. doi: 10.1093/nar/18.9.2801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Liu L. F., Rowe T. C., Yang L., Tewey K. M., Chen G. L. Cleavage of DNA by mammalian DNA topoisomerase II. J Biol Chem. 1983 Dec 25;258(24):15365–15370. [PubMed] [Google Scholar]
  24. Liu L. F., Wang J. C. DNA-DNA gyrase complex: the wrapping of the DNA duplex outside the enzyme. Cell. 1978 Nov;15(3):979–984. doi: 10.1016/0092-8674(78)90281-7. [DOI] [PubMed] [Google Scholar]
  25. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  26. Maxwell A., Gellert M. Mechanistic aspects of DNA topoisomerases. Adv Protein Chem. 1986;38:69–107. doi: 10.1016/s0065-3233(08)60526-4. [DOI] [PubMed] [Google Scholar]
  27. Morrison A., Cozzarelli N. R. Site-specific cleavage of DNA by E. coli DNA gyrase. Cell. 1979 May;17(1):175–184. doi: 10.1016/0092-8674(79)90305-2. [DOI] [PubMed] [Google Scholar]
  28. Pato M. L., Howe M. M., Higgins N. P. A DNA gyrase-binding site at the center of the bacteriophage Mu genome is required for efficient replicative transposition. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8716–8720. doi: 10.1073/pnas.87.22.8716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Shuman S., Prescott J. Specific DNA cleavage and binding by vaccinia virus DNA topoisomerase I. J Biol Chem. 1990 Oct 15;265(29):17826–17836. [PubMed] [Google Scholar]
  30. Svejstrup J. Q., Christiansen K., Gromova I. I., Andersen A. H., Westergaard O. New technique for uncoupling the cleavage and religation reactions of eukaryotic topoisomerase I. The mode of action of camptothecin at a specific recognition site. J Mol Biol. 1991 Dec 5;222(3):669–678. doi: 10.1016/0022-2836(91)90503-x. [DOI] [PubMed] [Google Scholar]
  31. Thomsen B., Mollerup S., Bonven B. J., Frank R., Blöcker H., Nielsen O. F., Westergaard O. Sequence specificity of DNA topoisomerase I in the presence and absence of camptothecin. EMBO J. 1987 Jun;6(6):1817–1823. doi: 10.1002/j.1460-2075.1987.tb02436.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Tse-Dinh Y. C., McCarron B. G., Arentzen R., Chowdhry V. Mechanistic study of E. coli DNA topoisomerase I: cleavage of oligonucleotides. Nucleic Acids Res. 1983 Dec 20;11(24):8691–8701. doi: 10.1093/nar/11.24.8691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tse Y. C., Kirkegaard K., Wang J. C. Covalent bonds between protein and DNA. Formation of phosphotyrosine linkage between certain DNA topoisomerases and DNA. J Biol Chem. 1980 Jun 25;255(12):5560–5565. [PubMed] [Google Scholar]
  34. Vershon A. K., Liao S. M., McClure W. R., Sauer R. T. Interaction of the bacteriophage P22 Arc repressor with operator DNA. J Mol Biol. 1987 May 20;195(2):323–331. doi: 10.1016/0022-2836(87)90653-x. [DOI] [PubMed] [Google Scholar]
  35. Vipond I. B., Baldwin G. S., Halford S. E. Divalent metal ions at the active sites of the EcoRV and EcoRI restriction endonucleases. Biochemistry. 1995 Jan 17;34(2):697–704. doi: 10.1021/bi00002a037. [DOI] [PubMed] [Google Scholar]
  36. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]
  37. Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1996;65:635–692. doi: 10.1146/annurev.bi.65.070196.003223. [DOI] [PubMed] [Google Scholar]
  38. Young D. B., Cole S. T. Leprosy, tuberculosis, and the new genetics. J Bacteriol. 1993 Jan;175(1):1–6. doi: 10.1128/jb.175.1.1-6.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang H. L., DiGate R. J. The carboxyl-terminal residues of Escherichia coli DNA topoisomerase III are involved in substrate binding. J Biol Chem. 1994 Mar 25;269(12):9052–9059. [PubMed] [Google Scholar]
  40. Zhu C. X., Roche C. J., Tse-Dinh Y. C. Effect of Mg(II) binding on the structure and activity of Escherichia coli DNA topoisomerase I. J Biol Chem. 1997 Jun 27;272(26):16206–16210. doi: 10.1074/jbc.272.26.16206. [DOI] [PubMed] [Google Scholar]

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