Abstract
The eukaryotic topoisomerase I (topo I) is the target of the cytotoxic alkaloid camptothecin (CTT). In vitro, CTT enhances the breakage of DNA by topo I when the reaction is stopped with detergent. Although breakage at some sites is enhanced to a great extent while breakage at others is enhanced only minimally, CTT does not significantly change the breakage specificity of topo I in vitro. It has been suggested that CTT acts by slowing the reclosure step of the nicking-closing reaction. To test this hypothesis, we have measured the rate of reclosure for different break sites in the presence of CTT after adding 0.5 M NaCl to a standard low salt reaction. In support of the hypothesis, we find that topo I-mediated DNA breakage is enhanced the greatest at those sites where closure of the break is the slowest. These results suggest a mechanism for the toxicity of CTT in vivo.
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Selected References
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- Abelson H. T., Penman S. Induction of alkali labile links in cellular DNA by camptothecin. Biochem Biophys Res Commun. 1973 Feb 20;50(4):1048–1054. doi: 10.1016/0006-291x(73)91512-x. [DOI] [PubMed] [Google Scholar]
- Abelson H. T., Penman S. Selective interruption of high molecular weight RNA synthesis in HeLa cells by camptothecin. Nat New Biol. 1972 May 31;237(74):144–146. doi: 10.1038/newbio237144a0. [DOI] [PubMed] [Google Scholar]
- Avemann K., Knippers R., Koller T., Sogo J. M. Camptothecin, a specific inhibitor of type I DNA topoisomerase, induces DNA breakage at replication forks. Mol Cell Biol. 1988 Aug;8(8):3026–3034. doi: 10.1128/mcb.8.8.3026. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Been M. D., Burgess R. R., Champoux J. J. Nucleotide sequence preference at rat liver and wheat germ type 1 DNA topoisomerase breakage sites in duplex SV40 DNA. Nucleic Acids Res. 1984 Apr 11;12(7):3097–3114. doi: 10.1093/nar/12.7.3097. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Champoux J. J., Aronoff R. The effects of camptothecin on the reaction and the specificity of the wheat germ type I topoisomerase. J Biol Chem. 1989 Jan 15;264(2):1010–1015. [PubMed] [Google Scholar]
- Champoux J. J. DNA is linked to the rat liver DNA nicking-closing enzyme by a phosphodiester bond to tyrosine. J Biol Chem. 1981 May 25;256(10):4805–4809. [PubMed] [Google Scholar]
- Champoux J. J. Evidence for an intermediate with a single-strand break in the reaction catalyzed by the DNA untwisting enzyme. Proc Natl Acad Sci U S A. 1976 Oct;73(10):3488–3491. doi: 10.1073/pnas.73.10.3488. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Champoux J. J., McConaughy B. L. Priming of superhelical SV40 DNA by Escherichia coli RNA polymerase for in vitro DNA synthesis. Biochemistry. 1975 Jan 28;14(2):307–316. doi: 10.1021/bi00673a017. [DOI] [PubMed] [Google Scholar]
- Champoux J. J. Mechanism of the reaction catalyzed by the DNA untwisting enzyme: attachment of the enzyme to 3'-terminus of the nicked DNA. J Mol Biol. 1978 Jan 25;118(3):441–446. doi: 10.1016/0022-2836(78)90238-3. [DOI] [PubMed] [Google Scholar]
- Champoux J. J. Strand breakage by the DNA untwisting enzyme results in covalent attachment of the enzyme to DNA. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3800–3804. doi: 10.1073/pnas.74.9.3800. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Champoux J. J. Topoisomerase I is preferentially associated with isolated replicating simian virus 40 molecules after treatment of infected cells with camptothecin. J Virol. 1988 Oct;62(10):3675–3683. doi: 10.1128/jvi.62.10.3675-3683.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eng W. K., Faucette L., Johnson R. K., Sternglanz R. Evidence that DNA topoisomerase I is necessary for the cytotoxic effects of camptothecin. Mol Pharmacol. 1988 Dec;34(6):755–760. [PubMed] [Google Scholar]
- Gellert M. DNA topoisomerases. Annu Rev Biochem. 1981;50:879–910. doi: 10.1146/annurev.bi.50.070181.004311. [DOI] [PubMed] [Google Scholar]
- Gilmour D. S., Elgin S. C. Localization of specific topoisomerase I interactions within the transcribed region of active heat shock genes by using the inhibitor camptothecin. Mol Cell Biol. 1987 Jan;7(1):141–148. doi: 10.1128/mcb.7.1.141. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hertzberg R. P., Dervan P. B. Cleavage of DNA with methidiumpropyl-EDTA-iron(II): reaction conditions and product analyses. Biochemistry. 1984 Aug 14;23(17):3934–3945. doi: 10.1021/bi00312a022. [DOI] [PubMed] [Google Scholar]
- Horwitz S. B., Chang C. K., Grollman A. P. Studies on camptothecin. I. Effects of nucleic acid and protein synthesis. Mol Pharmacol. 1971 Nov;7(6):632–644. [PubMed] [Google Scholar]
- Hsiang Y. H., Hertzberg R., Hecht S., Liu L. F. Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J Biol Chem. 1985 Nov 25;260(27):14873–14878. [PubMed] [Google Scholar]
- Jaxel C., Kohn K. W., Pommier Y. Topoisomerase I interaction with SV40 DNA in the presence and absence of camptothecin. Nucleic Acids Res. 1988 Dec 9;16(23):11157–11170. doi: 10.1093/nar/16.23.11157. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kjeldsen E., Bonven B. J., Andoh T., Ishii K., Okada K., Bolund L., Westergaard O. Characterization of a camptothecin-resistant human DNA topoisomerase I. J Biol Chem. 1988 Mar 15;263(8):3912–3916. [PubMed] [Google Scholar]
- Kjeldsen E., Mollerup S., Thomsen B., Bonven B. J., Bolund L., Westergaard O. Sequence-dependent effect of camptothecin on human topoisomerase I DNA cleavage. J Mol Biol. 1988 Jul 20;202(2):333–342. doi: 10.1016/0022-2836(88)90462-7. [DOI] [PubMed] [Google Scholar]
- Martin S. R., McCoubrey W. K., Jr, McConaughy B. L., Young L. S., Been M. D., Brewer B. J., Champoux J. J. Multiple forms of rat liver type I topoisomerase. Methods Enzymol. 1983;100:137–144. doi: 10.1016/0076-6879(83)00050-6. [DOI] [PubMed] [Google Scholar]
- 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]
- 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]
- McConaughy B. L., Young L. S., Champoux J. J. The effect of salt on the binding of the eucaryotic DNA nicking-closing enzyme to DNA and chromatin. Biochim Biophys Acta. 1981 Aug 27;655(1):1–8. doi: 10.1016/0005-2787(81)90059-9. [DOI] [PubMed] [Google Scholar]
- Nitiss J., Wang J. C. DNA topoisomerase-targeting antitumor drugs can be studied in yeast. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7501–7505. doi: 10.1073/pnas.85.20.7501. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Perez-Stable C., Shen C. C., Shen C. K. Enrichment and depletion of Hela topoisomerase I recognition sites among specific types of DNA elements. Nucleic Acids Res. 1988 Aug 25;16(16):7975–7993. doi: 10.1093/nar/16.16.7975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Porter S. E., Champoux J. J. Mapping in vivo topoisomerase I sites on simian virus 40 DNA: asymmetric distribution of sites on replicating molecules. Mol Cell Biol. 1989 Feb;9(2):541–550. doi: 10.1128/mcb.9.2.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Snapka R. M., Powelson M. A., Strayer J. M. Swiveling and decatenation of replicating simian virus 40 genomes in vivo. Mol Cell Biol. 1988 Feb;8(2):515–521. doi: 10.1128/mcb.8.2.515. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Snapka R. M. Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication. Mol Cell Biol. 1986 Dec;6(12):4221–4227. doi: 10.1128/mcb.6.12.4221. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Van Dyke M. W., Dervan P. B. Methidiumpropyl-EDTA.Fe(II) and DNase I footprinting report different small molecule binding site sizes on DNA. Nucleic Acids Res. 1983 Aug 25;11(16):5555–5567. doi: 10.1093/nar/11.16.5555. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang J. C. DNA topoisomerases. Annu Rev Biochem. 1985;54:665–697. doi: 10.1146/annurev.bi.54.070185.003313. [DOI] [PubMed] [Google Scholar]