A mutant yeast type II topoisomerase was generated by in vitro mutagenesis followed by selection in vivo for resistance to the quinolone CP-115,953. The resulting mutant enzyme had a single point mutation which converted His to Tyr (top2H1012Y). top2H1012Y was overexpressed in yeast, purified, and characterized in vitro. The mutant type II topoisomerase was slightly less active than the wild type enzyme, apparently due to a decreased affinity for DNA. The affinity of the mutant enzyme for ATP was similar to that of wild type topoisomerase II. As determined by DNA cleavage assays, top2H1012Y was resistant to CP-115,953 and etoposide both prior to and following the DNA strand-passage event. In marked contrast, the mutant enzyme displayed wild type sensitivity to amsacrine and was severalfold hypersensitive to ellipticine. A similar pattern of resistance was observed in yeast cells harboring the top2H1012Y allele. Thus, it appears that the mutant type II topoisomerase can distinguish between nonintercalative and intercalative agents. Finally, the His Tyr mutation defines a potential new drug resistance-conferring region on eukaryotic topoisomerase II.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				A. T. Rogojina and J. L. Nitiss Isolation and Characterization of mAMSA-hypersensitive Mutants: CYTOTOXICITY OF Top2 COVALENT COMPLEXES CONTAINING DNA SINGLE STRAND BREAKS J. Biol. Chem., October 24, 2008; 283(43): 29239 - 29250. [Abstract] [Full Text] [PDF]

				S. K. Hasan, A. N. Mays, T. Ottone, A. Ledda, G. La Nasa, C. Cattaneo, E. Borlenghi, L. Melillo, E. Montefusco, J. Cervera, et al. Molecular analysis of t(15;17) genomic breakpoints in secondary acute promyelocytic leukemia arising after treatment of multiple sclerosis Blood, October 15, 2008; 112(8): 3383 - 3390. [Abstract] [Full Text] [PDF]

				J. E. Deweese, A. B. Burgin, and N. Osheroff Human topoisomerase II{alpha} uses a two-metal-ion mechanism for DNA cleavage Nucleic Acids Res., September 1, 2008; 36(15): 4883 - 4893. [Abstract] [Full Text] [PDF]

				C. Leontiou, J. H. Lakey, and C. A. Austin Mutation E522K in Human DNA Topoisomerase II{beta} Confers Resistance to Methyl N-(4'-(9-acridinylamino)-phenyl)carbamate hydrochloride and Methyl N-(4'-(9-acridinylamino)-3-methoxy-phenyl) methane sulfonamide but Hypersensitivity to Etoposide Mol. Pharmacol., September 1, 2004; 66(3): 430 - 439. [Abstract] [Full Text] [PDF]

				J. V. Walker, K. C. Nitiss, L. H. Jensen, C. Mayne, T. Hu, P. B. Jensen, M. Sehested, T. Hsieh, and J. L. Nitiss A Mutation in Human Topoisomerase II {alpha} Whose Expression Is Lethal in DNA Repair-deficient Yeast Cells J. Biol. Chem., June 18, 2004; 279(25): 25947 - 25954. [Abstract] [Full Text] [PDF]

				D. S. Sappal, A. K. McClendon, J. A. Fleming, V. Thoroddsen, K. Connolly, C. Reimer, R. K. Blackman, C. E. Bulawa, N. Osheroff, P. Charlton, et al. Biological characterization of MLN944: A potent DNA binding agent Mol. Cancer Ther., January 1, 2004; 3(1): 47 - 58. [Abstract] [Full Text]

				C. I. Lasmezas Putative functions of PrPC Br. Med. Bull., June 1, 2003; 66(1): 61 - 70. [Abstract] [Full Text] [PDF]

				D. Ince, X. Zhang, L. C. Silver, and D. C. Hooper Dual Targeting of DNA Gyrase and Topoisomerase IV: Target Interactions of Garenoxacin (BMS-284756, T-3811ME), a New Desfluoroquinolone Antimicrob. Agents Chemother., November 1, 2002; 46(11): 3370 - 3380. [Abstract] [Full Text] [PDF]

				D. Strumberg, J. L. Nitiss, J. Dong, J. Walker, M. C. Nicklaus, K. W. Kohn, J. G. Heddle, A. Maxwell, S. Seeber, and Y. Pommier Importance of the Fourth Alpha-Helix within the CAP Homology Domain of Type II Topoisomerase for DNA Cleavage Site Recognition and Quinolone Action Antimicrob. Agents Chemother., September 1, 2002; 46(9): 2735 - 2746. [Abstract] [Full Text] [PDF]

				S. I. Khan, A. C. Nimrod, M. Mehrpooya, J. L. Nitiss, L. A. Walker, and A. M. Clark Antifungal Activity of Eupolauridine and Its Action on DNA Topoisomerases Antimicrob. Agents Chemother., June 1, 2002; 46(6): 1785 - 1792. [Abstract] [Full Text] [PDF]

				N. F. KRYNETSKAIA, X. CAI, J. L. NITISS, E. Y. KRYNETSKI, and M. V. RELLING Thioguanine substitution alters DNA cleavage mediated by topoisomerase II FASEB J, November 1, 2000; 14(14): 2339 - 2344. [Abstract] [Full Text]

				J. Dong, J. Walker, and J. L. Nitiss A Mutation in Yeast Topoisomerase II That Confers Hypersensitivity to Multiple Classes of Topoisomerase II Poisons J. Biol. Chem., March 10, 2000; 275(11): 7980 - 7987. [Abstract] [Full Text] [PDF]

				L. H. Jensen, K. C. Nitiss, A. Rose, J. Dong, J. Zhou, T. Hu, N. Osheroff, P. B. Jensen, M. Sehested, and J. L. Nitiss A Novel Mechanism of Cell Killing by Anti-topoisomerase II Bisdioxopiperazines J. Biol. Chem., January 21, 2000; 275(3): 2137 - 2146. [Abstract] [Full Text] [PDF]

				V. E. Anderson, R. P. Zaniewski, F. S. Kaczmarek, T. D. Gootz, and N. Osheroff Quinolones Inhibit DNA Religation Mediated by Staphylococcus aureus Topoisomerase IV. CHANGES IN DRUG MECHANISM ACROSS EVOLUTIONARY BOUNDARIES J. Biol. Chem., December 10, 1999; 274(50): 35927 - 35932. [Abstract] [Full Text] [PDF]

				D. Strumberg, J. L. Nitiss, J. Dong, K. W. Kohn, and Y. Pommier Molecular Analysis of Yeast and Human Type II Topoisomerases. ENZYME-DNA AND DRUG INTERACTIONS J. Biol. Chem., October 1, 1999; 274(40): 28246 - 28255. [Abstract] [Full Text] [PDF]

				D. Strumberg, J. L. Nitiss, A. Rose, M. C. Nicklaus, and Y. Pommier Mutation of a Conserved Serine Residue in a Quinolone-resistant Type II Topoisomerase Alters the Enzyme-DNA and Drug Interactions J. Biol. Chem., March 12, 1999; 274(11): 7292 - 7301. [Abstract] [Full Text] [PDF]

				D. A. Burden and N. Osheroff In Vitro Evolution of Preferred Topoisomerase II DNA Cleavage Sites J. Biol. Chem., February 19, 1999; 274(8): 5227 - 5235. [Abstract] [Full Text] [PDF]

				M. Sabourin, J. A. W. Byl, S. E. Hannah, J. L. Nitiss, and N. Osheroff A Mutant Yeast Topoisomerase II (top2G437S) with Differential Sensitivity to Anticancer Drugs in the Presence and Absence of ATP J. Biol. Chem., October 30, 1998; 273(44): 29086 - 29092. [Abstract] [Full Text] [PDF]

				J. M. Fortune and N. Osheroff Merbarone Inhibits the Catalytic Activity of Human Topoisomerase IIalpha by Blocking DNA Cleavage J. Biol. Chem., July 10, 1998; 273(28): 17643 - 17650. [Abstract] [Full Text] [PDF]

				D. A. Burden, P. S. Kingma, S. J. Froelich-Ammon, M.-A. Bjornsti, M. W. Patchan, R. B. Thompson, and N. Osheroff Topoisomerase II·Etoposide Interactions Direct the Formation of Drug-induced Enzyme-DNA Cleavage Complexes J. Biol. Chem., November 15, 1996; 271(46): 29238 - 29244. [Abstract] [Full Text] [PDF]

				J. Sekiguchi, J. T. Stivers, A. S. Mildvan, and S. Shuman Mechanism of Inhibition of Vaccinia DNA Topoisomerase by Novobiocin and Coumermycin J. Biol. Chem., January 26, 1996; 271(4): 2313 - 2322. [Abstract] [Full Text] [PDF]

				S. J. Froelich-Ammon, D. A. Burden, M. W. Patchan, S. H. Elsea, R. B. Thompson, and N. Osheroff Increased Drug Affinity as the Mechanistic Basis for Drug Hypersensitivity of a Mutant Type II Topoisomerase J. Biol. Chem., November 24, 1995; 270(47): 28018 - 28021. [Abstract] [Full Text] [PDF]

				S. J. Froelich-Ammon and N. Osheroff Topoisomerase Poisons: Harnessing the Dark Side of Enzyme Mechanism J. Biol. Chem., September 15, 1995; 270(37): 21429 - 21432. [Full Text] [PDF]

				Y. Hsiung, S. H. Elsea, N. Osheroff, and J. L. Nitiss A Mutation in Yeast TOP2 Homologous to a Quinolone-resistant Mutation in Bacteria J. Biol. Chem., September 1, 1995; 270(35): 20359 - 20364. [Abstract] [Full Text] [PDF]

				S. J. Froelich-Ammon, M. W. Patchan, N. Osheroff, and R. B. Thompson Topoisomerase II Binds to Ellipticine in the Absence or Presence of DNA J. Biol. Chem., June 23, 1995; 270(25): 14998 - 15004. [Abstract] [Full Text] [PDF]

				J. M. Fortune, O. V. Lavrukhin, J. R. Gurnon, J. L. Van Etten, R. S. Lloyd, and N. Osheroff Topoisomerase II from Chlorella Virus PBCV-1 Has an Exceptionally High DNA Cleavage Activity J. Biol. Chem., June 22, 2001; 276(26): 24401 - 24408. [Abstract] [Full Text] [PDF]