Mms22p protects Saccharomyces cerevisiae from DNA damage induced by topoisomerase II

Abstract

The cleavage reaction of topoisomerase II, which creates double-stranded DNA breaks, plays a central role in both the cure and initiation of cancer. Therefore, it is important to understand the cellular processes that repair topoisomerase II-generated DNA damage. Using a genome-wide approach with Saccharomyces cerevisiae, we found that Deltamre11, Deltaxrs2, Deltarad50, Deltarad51, Deltarad52, Deltarad54, Deltarad55, Deltarad57 and Deltamms22 strains were hypersensitive to etoposide, a drug that specifically increases levels of topoisomerase II-mediated DNA breaks. These results confirm that the single-strand invasion pathway of homologous recombination is the major pathway that repairs topoisomerase II-induced DNA damage in yeast and also indicate an important role for Mms22p. Although Deltamms22 strains are sensitive to several DNA-damaging agents, little is known about the function of Mms22p. Deltamms22 cultures accumulate in G2/M, and display an abnormal cell cycle response to topoisomerase II-mediated DNA damage. MMS22 appears to function outside of the single-strand invasion pathway, but levels of etoposide-induced homologous recombination in Deltamms22 cells are lower than wild-type. MMS22 is epistatic with RTT101 and RTT107, genes that encode its protein binding partners. Finally, consistent with a role in DNA processes, Mms22p localizes to discrete nuclear foci, even in the absence of etoposide or its binding partners.

Figure 1

Pathways used to repair double-stranded DNA breaks in S.cerevisiae. Components of the pathways that play integral roles in homologous recombination and nonhomologous end-joining are shown. A previous study that used deletion mutants in these pathways suggested that the single-strand invasion pathway of homologous recombination is primarily responsible for repairing topoisomerase II-generated DNA breaks that are stabilized by etoposide (34).

Figure 2

Genes involved in protecting cells from etoposide-induced DNA damage. A S.cerevisiae haploid deletion library (∼4800 strains) was screened for sensitivity to etoposide. Wild-type (WT, BY4741) and indicated deletion strains were plated in serial dilution onto YPD medium containing drug solvent (DMSO) or 1 mM etoposide.

Figure 3

Δmms22 cells are hypersensitive to etoposide and amsacrine. The sensitivity of Δmms22 to topoisomerase II poisons was tested. Serial dilutions of wild-type (WT), Δmms22 and Δrad52 cultures were plated onto YPD medium containing DMSO or 100 μM drug (top). Cytotoxicity assays were performed using the indicated strains. Cells were exposed to etoposide (bottom, left panel) or amsacrine (bottom, right panel) for 8 h in liquid culture. Error bars represent the SD values of at least three independent experiments.

Figure 4

Expression of plasmid-encoded MMS22 rescues the Δmms22 drug hypersensitive phenotype. MMS22 was cloned, along with its endogenous promoter, into pRS416 to generate pMMS22. Serial dilutions of the wild-type strain carrying pRS416 (empty vector) as well as the Δmms22 strain carrying either pRS416 or pMMS22 were plated onto SC-URA medium containing DMSO or 100 μM etoposide (top). Cytotoxicity assays were performed using the indicated strains (bottom). Cells were exposed to etoposide for 8 h in liquid culture. Error bars represent the SD values of at least three independent experiments.

Figure 5

Δmms22 cells accumulate in G₂/M. Asynchronous wild-type and Δmms22 cells were grown for 6 h in the presence of DMSO (black line) or 50 μM etoposide (red line). Peaks representing haploid (1 N) and diploid (2 N) DNA contents are indicated (top). The percent of cultures in G₀/G₁, S or G₂/M phase are shown (bottom). Cells were analyzed with Sytox Green as the DNA stain. Results are representative of three independent experiments.

Figure 6

MMS22 is not epistatic to RAD54. A Δmms22 Δrad54 double mutant was constructed to determine whether Mms22p is involved in the single-strand invasion pathway of homologous recombination. Wild-type, Δmms22, Δrad54 and Δmms22 Δrad54 double mutant cells were plated in serial dilution onto YPD medium containing DMSO or 50 μM etoposide (top). Cytotoxicity assays were performed using the indicated strains (bottom). Cells were exposed to etoposide for 24 h in liquid culture. Error bars represent the SD values of at least three independent experiments.

Figure 7

Etoposide-induced homologous recombination is lower in the Δmms22 strain. A plasmid-based (YCpHR) reporter assay (34,38) was used to assess levels of homologous recombination in yeast. A strain expressing allelic top2S740W was utilized in these studies, since this mutant topoisomerase II is hypersensitive to etoposide. Wild-type and Δmms22 cells containing the top2S740W allele were exposed to etoposide for 5 h. Error bars represent the SD values of four independent experiments.

Figure 8

MMS22 is epistatic to RTT101 and RTT107. Wild-type (WT); Δmms22, Δrtt101 and Δrtt107 single mutant; Δmms22 Δrtt101, Δmms22 Δrtt107 and Δrtt101 Δrtt107 double mutant; and Δmms22 Δrtt101 Δrtt107 triple mutant cells were plated in serial dilution onto YPD medium containing DMSO or 50 μM etoposide (top). Cytotoxicity assays were performed using the indicated strains (bottom). Cells were exposed to etoposide for 24 h in liquid culture. Error bars represent the SD values of at least three independent experiments.

Figure 9

GFP-Mms22p localizes to nuclear foci. Δmms22 cells containing a vector (pGFP-N-FUS) that expressed GFP or an N-terminal GFP-MMS22 fusion construct (pGFP-MMS22) were examined for hypersensitivity to etoposide to confirm that the GFP-Mms22p fusion protein was functional. Wild-type (WT) cells carrying pGFP-N-FUS as well as Δmms22 cells carrying either pGFP-N-FUS or pGFP-MMS22 were plated in serial dilution onto SC-MET/URA medium containing DMSO or 100 μM etoposide (top). GFP and the GFP-Mms22p fusion protein were visualized in cells by direct fluorescence microscopy. DNA was localized by Hoechst staining. Differential image contrast (DIC) images of the visualized yeast cells are shown for reference.