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. 2003 Aug 1;31(15):4373-84.
doi: 10.1093/nar/gkg497.

Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks

Michelle Sabourin  1 , John L Nitiss, Karin C Nitiss, Kazuo Tatebayashi, Hideo Ikeda, Neil Osheroff
Affiliations

Yeast recombination pathways triggered by topoisomerase II-mediated DNA breaks

Michelle Sabourin et al. Nucleic Acids Res. .

Abstract

Topoisomerase II is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating transient double-strand DNA breaks. While the enzyme cannot perform its essential cellular functions without cleaving DNA, this scission activity is inherently dangerous to chromosomal integrity. In fact, etoposide and other clinically important anticancer drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. Cells rely heavily on recombination to repair double-strand DNA breaks, but the specific pathways used to repair topoisomerase II-generated DNA damage have not been defined. Therefore, Saccharomyces cerevisiae was used as a model system to delineate the recombination pathways that repair DNA breaks generated by topoisomerase II. Yeast cells that expressed wild-type or a drug-hypersensitive mutant topoisomerase II or overexpressed the wild-type enzyme were examined. Based on cytotoxicity and recombination induced by etoposide in different repair-deficient genetic backgrounds, double-strand DNA breaks generated by topoisomerase II appear to be repaired primarily by the single-strand invasion pathway of homologous recombination. Non-homologous end joining also was triggered by etoposide treatment, but this pathway was considerably less active than single-strand invasion and did not contribute significantly to cell survival in S.cerevisiae.

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Figures

Figure 1
Figure 1
Pathways of double-strand DNA break repair in S.cerevisiae. Components that play integral roles in homologous recombination (single-strand invasion and single-strand annealing) and non-homologous end joining pathways are shown.
Figure 2
Figure 2
Cytotoxicity of etoposide to repair-compromised yeast strains. The effects of etoposide on cell survival of yeast deficient in a series of repair genes are shown. The mutations were made in three different strain backgrounds: the original strain which expressed wild-type TOP2 (TOP2) (A), a mutant strain in which the chromosomal copy of TOP2 was replaced with the etoposide hypersensitive top2S740W allele (top2S740W) (B) and the TOP2 strain which overexpressed wild-type TOP2 from the plasmid pDED1TOP2 (pDED1TOP2) (C). The mutations introduced in these strains were Δrad1 (open squares), Δku70 (closed circles), Δrad54 (open circles), Δrad50 (closed triangles) and Δrad52 (open triangles). Repair-proficient parental strains (parental) are represented by closed squares. Data were calculated relative to cell growth at 24 h in drug-free cultures, such that the level of growth for each strain in the absence of etoposide was set to 100%. Data represent the average of two or more independent experiments, each plated in duplicate.
Figure 3
Figure 3
Restriction digests of intact and recombined YCpHR homologous recombination reporter plasmids. A map of the homologous recombination reporter plasmid YCpHR is shown at the top. The four black lines along the backbone of the plasmid indicate PstI cut sites and the numbers internal to the plasmid correspond to the restriction fragment sizes as indicated on the gel at the bottom. Plasmids were rescued from TOP2 strains before (WT) and after (R1, R2 and R3) exposure to etoposide, CP-115,953 or camptothecin, respectively. Samples were digested with PstI and subjected to electrophoresis in an agarose gel that contained ethidium bromide. A corresponding digest of the original plasmid is also shown (Control). The sizes of restriction digest fragments are indicated to the left in kilobases. The restriction fragment representing the recombined portion of YCpHR is indicated at the right (*).
Figure 4
Figure 4
Homologous recombination in YCpHR triggered by drugs. Repair-proficient yeast expressing either wild-type (TOP2, closed bars) or mutant (top2S740W, open bars) topoisomerase II transformed with YCpHR were treated with 170 µM etoposide, 180 µM TOP-53, 50 µM CP-115,953 or 140 µM camptothecin for 5 h. Data represent the number of recombinant cells per 104 viable cells and are the average of two or more independent experiments, each plated in duplicate. Standard deviations are indicated by error bars. (Inset) Levels of etoposide-triggered homologous recombination in YCpHR in repair-proficient yeast that also carried the topoisomerase II overexpression plasmid (pDED1TOP2).
Figure 5
Figure 5
Homologous recombination in YCpHR triggered by topoisomerase II-generated DNA damage. Repair-deficient strains carrying the YCpHR reporter plasmid were exposed to 0–170 µM etoposide for 5 h. Data represent the number of recombinant cells per 104 viable cells. Repair-deficient strains generated in the TOP2 and top2S740W backgrounds are shown in (A) and (B), respectively. Strains are labeled as in Figure 2. Data generated at 0 and 170 µM etoposide for the TOP2, top2S740W and pDED1TOP2 (which overexpresses topoisomerase II) strains are shown in (C) (standard errors of the mean are represented by error bars). Data represent the average of two or more independent experiments, each plated in duplicate.
Figure 6
Figure 6
Non-homologous end joining in YCpL2 triggered by topoisomerase II-generated DNA damage. The repair-proficient top2S740W strain was transformed with YCpL2, a reporter plasmid related to YCpHR but used to monitor non-homologous end joining. Cultures were treated with 0–170 µM etoposide for 5 h. Data represent the number of recombinant cells per 104 viable cells and are the average of two independent experiments plated in duplicate. Standard errors of the mean are represented by error bars. A map of YCpL2 is shown (not to scale).
Figure 7
Figure 7
Terminal phenotype of TOP2 strains in the presence of etoposide. The following yeast strains were grown for 24 h in the presence of 170 µM etoposide (green) or an equivalent amount of drug solvent (DMSO, red): TOP2 (A), TOP2Δrad1 (B) and TOP2Δrad52 (C). Peaks representing haploid (1N) and diploid (2N) DNA contents are indicated. Aliquots of 1 ml were removed and used for FACS analysis with Sytox Green as the DNA stain.
Figure 8
Figure 8
Cell cycle progression of yeast during exposure to etoposide. The indicated strains were grown for 24 h in the absence of drug (TOP2/no drug) (A) or in the presence of 170 µM etoposide as follows: TOP2/Δrad1 (B), TOP2 (C), TOP2/Δrad52 (D), top2S740W (E), top2S740W/Δrad52 (F), pDED1TOP2 (G), pDED1TOP2/Δrad52 (H). Aliquots of 1 ml were removed every 1.5 h from 0–9 h and used for FACS analysis with Sytox Green as the DNA stain.
Figure 9
Figure 9
Effects of etoposide on the growth of yeast. Simultaneous to taking samples for the FACS analyses shown in Figure 8, yeast were diluted and plated in duplicate to YPDA to determine the number of viable cells at each time point. Cell growth is plotted relative to the number of cells at 0 h. Cultures were grown in the absence of drug (TOP2/no drug, closed squares) or in the presence of 170 µM etoposide as follows: TOP2/Δrad1 (open squares), TOP2 (closed circles), TOP2/Δrad52 (open circles), top2S740W (closed triangles), top2S740W/Δrad52 (open triangles), pDED1TOP2 (closed diamonds), pDED1TOP2/Δrad52 (open diamonds).
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References

    1. Wang J.C. (1996) DNA topoisomerases. Annu. Rev. Biochem., 65, 635–692. - PubMed
    1. Burden D.A. and Osheroff,N. (1998) Mechanism of action of eukaryotic topoisomerase II and drugs targeted to the enzyme. Biochim. Biophys. Acta, 1400, 139–154. - PubMed
    1. Nitiss J.L. (1998) Investigating the biological functions of DNA topoisomerases in eukaryotic cells. Biochim. Biophys. Acta, 1400, 63–81. - PubMed
    1. Wang J.C. (1998) Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine. Q. Rev. Biophys., 31, 107–144. - PubMed
    1. Fortune J.M. and Osheroff,N. (2000) Topoisomerase II as a target for anticancer drugs: when enzymes stop being nice. Prog. Nucleic Acid Res. Mol. Biol., 64, 221–253. - PubMed

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