We report the development of a rapid method to detect binding of supercoiled DNA to Escherichia coli topoisomerase I using the scintillation proximity assay (SPA). Streptavidin-SPA beads were coated with biotinylated topoisomerase I produced in vivo as a chimeric fusion protein. The hybrid biotinyl-fusion protein was overproduced in E. coli and purified in a single step by monomeric avidin affinity chromatography. The assay signal originates from both covalent and noncovalent binding of [3H]DNA to the SPA bead-immobilized enzyme. About 20-30% of the total [3H]DNA bound to the bead-immobilized enzyme remains associated with the bead in the presence of 0.5% SDS. The residual signal arises from the trapping of covalent [3H]DNA-enzyme complexes. The assay was employed in a high throughput screen that identified two general classes of topoisomerase inhibitors: agents that (1) inhibit DNA binding or (2) stabilize a covalent enzyme-DNA intermediate.
References
1.
Neu, H. C. (1992). The crisis in antibiotic resistance. Science257:1064.
2.
Hooper, D. C., Wolfson, J. S. (1991). Fluoroquinolone antimicrobial agents. N. Engl. J. Med.324:384.
3.
Luttinger, A. (1995). The twisted `life' of DNA in the cell: Bacterial topoisomerases. Mol. Microbiol.15:601.
4.
Sutcliffe, J. A., Gootz, T. D., Barrett, J. F. (1989). Biochemical characteristics and physiological significance of major DNA topoisomerases. Antimicrob. Agents Chemother. 33:2027.
5.
Wang, J. C. (1991). DNA topoisomerases: why so many?J. Biol. Chem.266:6659.
6.
Liu, L. F., Wang, J. C. (1987). Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. U.S.A.83:7024.
7.
Drolet, M., Bi, X., Liu, L. F. (1994). Hypernegative supercoiling of the DNA template during transcription elongation in vitro. J. Biol. Chem.269:2068.
8.
Drolet, M., Phoenix, P., Menzel, R., Massé, E., Liu, L. F., Crouch, R. J. (1995). Overexpression of RNase H partially complements the growth defect of an Escherichia coli topA mutant: R-loop formation is a major problem in the absence of DNA topoisomerase. I. Proc. Nat/. Acad. Sci. U.S.A.92:3526.
9.
DUrrenberger, M., Bjornsti, M.-A., Uetz, T., Hobot, J. A., Kellenberger, E. (1988). Intracellular location of the histone-like protein HU in Escherichia coli. J. Bacteriol.170:4757.
10.
Kaguni, J. M., Kornberg, A. (1984). Topoisomerase I confers specificity in enzymatic replication of the Escherichia coli chromosomal origin. J. Biol. Chem.259:8578.
11.
DiNardo, S., Voelkel, K. A., Sternglanz, R. (1982). Escherichia coli DNA topoisomerase I mutants have compensatory mutations in DNA gyrase genes. Cell31:43.
12.
Oram, M., Fischer, L. M. (1992). An Escherichia coli DNA topoisomerase I mutant has a compensatory mutation that alters two residues between functional domains of the DNA gyrase A protein. J. Bacteriol.174:4175.
13.
Pruss, G. J., Manes, S. H., Drlica, K. (1982). Escherichia coli DNA topoisomerase I mutants: Increased supercoiling is corrected by mutations near gyrase genes. Cell31:35.
14.
Raj, A., Xabel, D. J., Laufer, C. S., Depew, R. E. (1985). Genetic analysis of mutations that compensate for loss of Escherichia coli DNA topoisomerase I. J. Bacteriol.162:1173.
15.
Dorman, C. J., Lynch, A. S., Ni Bhriain, N., Higgins, C. F. (1989). DNA supercoiling in Escherichia coli: topA mutations can be suppressed by DNA amplifications involving the to/C locus. Mol. Microbiol.3:531.
16.
Kato, J., Nishimura, Y., Imamura, R., Niki, H., Hiraga, S., Suzuki, H. (1990). New topoisomerase essential for chromosome segregation in Escherichia coli. Cell63:393.
17.
Zechiedrich, E. L., Cozzarelli, N. R. (1995). Role of topoisomerase IV and DNA gyrase in DNA unlinking during replication in Escherichia coli. Genes Dev.9:2859.
18.
Galan, J. E., Curtiss, R. (1990). Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling. Infect. Immun.58:1879.
19.
Tse-Dinh, Y.-C., Beran-Steed, R. K. (1988). Excherichia coli DNA topoisomerase I is a zinc metalloprotein with three repetitive zinc-binding domains. J. Biol. Chem.263:15857.
20.
Sharma, A., Mondragón, A. (1995). DNA topoisomerases. Curr. Opin. Strict. Biol.5:39.
21.
Tse-Dinh, Y.-C. (1994). Biochemistry of bacterial type I DNA toposiomerases. Adv. Pharmacol.29A:21-37.
22.
Wang, J. C. (1994). DNA topoisomerases as targets of therapeutics: An overview. Adv. Pharmacol.29A:1-19.
23.
Wigley, D. B. (1995). Structure and mechanism of DNA topoisomerases. Annu. Rev. Biophys. Biomol. Strict.24:185.
24.
Mizushima, T., Natori, S., Sekimizu, K. (1992). Inhibition of Escherichia coli DNA topoisomerase I activity by phospholipids. Biochem. J.285:503.
25.
Tabary, X., Moreau, N., Dureuil, C., Le Goffic, F. (1987). Effect of DNA gyrase inhibitors pefloxacin, five other quinolones, novobiocin, and clorobiocin on Escherichia coli topoisomerase I. Antimicrob. Agents Chemother. 31:1925.
26.
Taylor, S. T., Menzel, R. (1995). The creation of a camptothecin-sensitive Escherichia coli based on the expression of the human topoisomerase I. Gene167:69.
27.
Froelich-Ammon, S. J., Osheroff, N. (1995). Topoisomerase poisons: Harnessing the dark side of enzyme mechanism. J. Biol. Chem.270:21429.
28.
Barrett, J. F., Sutcliffe, J. A., Gootz, T. D. (1989). In vitro assays used to measure the activity of topoisomerases. Antimicrob. Agents Chemother.34:1.
29.
Barrett, J. F., Bernstein, J. I., Krause, H. M., Hilliard, J. J., Ohemeng, K. A. (1993). Testing postential gyrase inhibitors of bacterial DNA gyrase: A comparison of the supercoiling inhibition assay and "cleavable complex' assay. Anal. Biochem.214:313.
30.
Bosworth, N., Towers, P. (1989). Scintillation proximity assay. Nature (London)341:167.
31.
Cronan, J. E. (1990). Biotination of proteins in vivo—a post-translational modification to label, purify, and study proteins. J. Biol. Chem.265:10327.
32.
Reed, K. E., Cronan, J. E. (1991). Escherichia coli exports previously folded and biotinated protein domains. J. Biol. Chem.266:11425.
33.
Wang, J. C., Becherer, K. (1983). Cloning of the gene topA encoding for DNA topoisomerase I and the physical mapping of the cysB-topA-trp region of Escherichia coli. Nucleic Acids Res.11:1773.
34.
Stacey, K. A., Simson, E. (1965). Improved method for the isolation of thymine-requiring mutants of Escherichia coli. J. Bacteriol.90:554.
35.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., Erlich, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science239:487.
36.
Sambrook, J., Fritsch, E. F., Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
37.
Kohanski, R. A., Lane, M. D. (1990). Monovalent avidin affinity columns. Methods Enzymol.184:194.
38.
Harlow, E., Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
39.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem.72:248.
40.
Eisman, E. R., Muller-Hill, B. (1990). Lac repressor forms stable loops in vitro with supercoiled wild-type lac DNA containing all three natural lac operators. J. Mol. Biol.213:763.
41.
Depew, R. E., Liu, L. F., Wang, J. C. (1978). Interaction between DNA and Escherichia coli protein w-formation of a complex between single-stranded DNA and w protein. J. Biol. Chem.253:511.
42.
Champoux, J. J. (1978). Mechanism of the reaction catalyzed by the DNA untwisting enzyme: Attachment of the enzyme to 3′-termnus of the nicked DNA. J. Mol. Biol.118:441.
43.
Madden, K. R., Stewart, L., Champoux, J. J. (1995). Preferential binding of human topoisomerase I to superhelical DNA. EMBO J.14:5399.
44.
Kirkegaard, K., Wang, J. C. (1985). Bacterial DNA topoisomerase I can relax positively supercoiled DNA containing a single-stranded loop. J. Mol. Biol.185:625.
45.
Lima, C. D., Wang, J. C., Mondragón, A. (1994). Three-dimensional structure of the 67K N-terminal fragment of E. coli DNA topoisomerase I. Nature (London)367:138.
46.
Roca, J. (1995). The mechanisms of DNA topoisomerases. Trends Biochem. Sci.20:156.
47.
Consler, T. G., Persson, B. L., Jung, H., Zen, K. H., Jung, K., Prive, G. G., Verner, G. E., Kaback, H. R. (1993). Properties and purification of an active biotinylated lactose permease from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A.90:6934.
