Effect of the DNA topoisomerase II inhibitor VP-16 on illegitimate recombination in yeast chromosomes.
Etoposide and teniposide, derivatives of podophyllotoxin, are inhibitors of DNA topoisomerase II and are potent anticancer agents. An adverse effect linked to the use of these drugs is the development of acute myeloid leukemia, a disorder usually associated with chromosomal translocation. To examine podophyllotoxin-induced DNA rearrangement, we developed an assay system to measure illegitimate recombination in Saccharomyces cerevisiae chromosomes. This approach uses juxtaposed CAN1-CYH2 negative selection markers that are introduced into the LEU2 locus, which is located on chromosome III, in a yeast strain carrying the mutated can1 and cyh2 genes. Upon formation of a deletion over the active CAN1-CYH2 genes, a cell becomes resistant to both canavanine and cycloheximide. To introduce drugs into the cell, we used a yeast strain carrying an ISE2 mutation, thereby making the cell drug-permeable. Here we show that treatment of cells with etoposide (VP-16) increases the rate of illegitimate recombination in yeast, indicating that VP-16 stimulates DNA topoisomerase-mediated illegitimate recombination. Structural analysis of the resulting recombinants indicate that most are formed by deletion mutations on chromosome III, which take place between short homologous regions of DNA. We propose a model for illegitimate recombination, in which VP-16 facilitates formation of a cleavable complex between DNA topoisomerase II and DNA, thus promoting DNA double-strand breakage with the resulting DNA ends joined by a non-homologous mechanism.
Asami
Yasuo
Y
Center for Basic Research, The Kitasato Institute, Minato-ku, Tokyo 108-8642, Japan.
Jia
Din-Wu
DW
Tatebayashi
Kazuo
K
Yamagata
Kazutsune
K
Tanokura
Masaru
M
Ikeda
Hideo
H
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics; D026905:Amino Acid Transport Systems / Q000235:genetics; D001483:Base Sequence; D015825:Chromosomes, Fungal / Q000235:genetics*; D005047:Etoposide / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics; D005819:Genetic Markers; D009154:Mutation; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D011995:Recombination, Genetic / Q000187:drug effects* / Q000235:genetics; D012269:Ribosomal Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics; D017384:Sequence Deletion; D059005:Topoisomerase II Inhibitors
D026905:Amino Acid Transport Systems; C097640:CAN1 protein, Candida albicans; D005656:Fungal Proteins; D005819:Genetic Markers; D019384:Nucleic Acid Synthesis Inhibitors; D012269:Ribosomal Proteins; D059005:Topoisomerase II Inhibitors; D005047:Etoposide; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase