Coordination of DNA damage responses via the Smc5/Smc6 complex - PubMed (original) (raw)

Coordination of DNA damage responses via the Smc5/Smc6 complex

Susan H Harvey et al. Mol Cell Biol. 2004 Jan.

Abstract

The detection of DNA damage activates DNA repair pathways and checkpoints to allow time for repair. Ultimately, these responses must be coordinated to ensure that cell cycle progression is halted until repair is completed. Several multiprotein complexes containing members of the structural maintenance of chromosomes family of proteins have been described, including the condensin and cohesin complexes, that are critical for chromosomal organization. Here we show that the Smc5/Smc6 (Smc5/6) complex is required for a coordinated response to DNA damage and normal chromosome integrity. Fission yeast cells lacking functional Smc6 initiate a normal checkpoint response to DNA damage, culminating in the phosphorylation and activation of the Chk1 protein kinase. Despite this, cells enter a lethal mitosis, presumably without completion of DNA repair. Another subunit of the complex, Nse1, is a conserved member of this complex and is also required for this response. We propose that the failure to maintain a checkpoint response stems from the lack of ongoing DNA repair or from defective chromosomal organization, which is the signal to maintain a checkpoint arrest. The Smc5/6 complex is fundamental to genome integrity and may function with the condensin and cohesin complexes in a coordinated manner.

PubMed Disclaimer

Figures

FIG. 1.

FIG. 1.

Overexpression of wild-type or dominant negative Rad18 leads to cell cycle arrest and a hollow-sphere-like nuclear phenotype. (A) Wild-type cells expressing vector, wild-type Rad18 (rad18+), or a dominant-negative form of Rad18 (rad18-dn) from the full-strength nmt1 promoter were grown in the absence of thiamine for 10 h to induce promoter derepression. Cell number was measured every 2 h for a subsequent 10 h. (B) DAPI-stained ethanol-fixed cells after 20 h of growth in the absence of thiamine were visualized by fluorescence microscopy. Note the cell elongation and large hollow-sphere-shaped nuclear staining visible after prolonged overexpression of either rad18+ or rad18-dn. (C) To determine Rad18 localization, GFP-tagged Rad18 was expressed from the wild-type nmt1 promoter. After approximately 8 h of promoter derepression (∼20 h of growth in medium lacking thiamine), the majority of GFP-Rad18 (green) does not colocalize with DNA (DAPI; red) in methanol-acetone-fixed cells. The same localization pattern was also observed for the GFP-tagged dominant-negative Rad18 (unpublished data). DIC, differential interference contrast; Merge, merged image of DAPI and GFP-Rad18 results.

FIG. 2.

FIG. 2.

The growth arrest and nuclear phenotypes of Rad18 overexpression are separable. To determine which domain of Rad18 produced the distinct hollow-sphere nuclear phenotype, various structural domains of Rad18 were overexpressed from the nmt1 promoter for 20 h in the absence of thiamine (corresponding to approximately 8 h of promoter derepression). Overexpression of full-length Rad18 produces a cell cycle delay, as evidenced by cell elongation; however, cell elongation was either not observed (N) or reduced (P) in the other constructs. Growth arrest was observed with overexpression of several different domains (−, no arrest; + to +++++, 20, 40, 60, 80, or 100% arrest compared to overexpression of full-length Rad18). The nuclear phenotypes are represented as the averages of at least three counts of 100 ethanol-fixed cells that had been stained with DAPI. Note that overexpression of amino acids 261 to 520 (the first coiled-coil domain) alone was sufficient to produce the nuclear-sphere phenotype.

FIG. 3.

FIG. 3.

A dominant-negative allele of rad18 is unable to maintain a DNA damage checkpoint arrest. Asynchronously growing cells expressing a vector control or rad18-dn from the nmt1 promoter were grown for 14 h in the absence of thiamine and irradiated with 100 J of UV-C/m2. (A) The kinetics of cell cycle arrest was measured by septation index analysis every 15 min for 2.5 h. (B) Abnormal mitoses were scored from ethanol-fixed cells stained with DAPI at the indicated times. Following UV-C irradiation, rad18-dn cells exhibited increased numbers of aberrant mitoses compared to the vector control cells. The data represent the means of three independent experiments. (C) DAPI-stained cells of the indicated strains exposed to 0 J of UV-C/m2 or at 150 min postexposure to 100 J of UV-C/m2. Arrows indicate cells undergoing abnormal mitoses. Bar, 10 μm.

FIG. 4.

FIG. 4.

Characterization of a conditional allele of rad18. A depletion allele of rad18 (designated rad18-so) was constructed by integrating a rad18 cDNA expressed from the weakest nmt promoter by sup3-5 integration into a rad18::ura4 background. (A) Northern blot of RNA extracted from wild-type (W) or rad18-so cells in the absence of thiamine (0 h) or after the indicated time in the presence of thiamine (1 to24 h) and probed with rad18. The results showed undetectable levels of rad18 mRNA after an hour of promoter repression (1 h). (B) Western blot analysis of the indicated strains (assayed as described for panel A) probed with anti-Rad18 antibodies. Note that the Rad18 protein remains detectable for several cell cycles after promoter repression (+T). (C and D) Relative cell numbers of wild-type and rad18-so cells grown in the presence and absence of thiamine over the indicated time period. Note that over the first 18 h of promoter repression the rad18-so strain exhibits relatively normal growth rates (C), while in the subsequent 18 h its growth rate is significantly reduced (D). (E) Ethanol-fixed rad18-so cells were stained with DAPI after 0 or 36 h in thiamine. After 36 h in thiamine, rad18-so cells displayed various nuclear abnormalities, including stretched and fragmented DNA and cells in which the nuclear material has been bisected by the septum, indicating mitotic failure. (F) FACS analysis of rad18-so cells at 0 or 36 h in thiamine shows the cells were arrested in G2 phase and mitosis.

FIG. 5.

FIG. 5.

rad18-so is unable to maintain an arrest in response to DNA damage. To deplete Rad18, thiamine was added to wild-type cells containing vector only or to nmtW -rad18-so cells. Mid-logarithmic cells were irradiated with 100 J of UV-C/m2 after 12.5 or 18 h of growth in thiamine (+T), which is prior to the appearance of cell cycle delay. (A) The kinetics of cell cycle arrest was measured using a septation index assay. (B) Abnormal mitoses were scored from DAPI-stained ethanol-fixed cells that showed high rates of abnormal mitoses in the rad18-so cells compared to those seen with control cells. The data represent the means of three independent experiments. (C) Ethanol-fixed cells were DAPI stained to assess the percentage of abnormal mitoses by fluorescence microscopy. The indicated strains were photographed in the absence of irradiation (0 J/m2) or at 150 min postir-radiation with 100 J of UV-C/m2 (100 J/m2). Note the predominance of stretched nuclear material and bisected cells, which comprise more than 80% of the total mitoses (∼25% of total cells) in rad18-so cells at 150 min post-UV irradiation. Bar, 10 μm.

FIG. 6.

FIG. 6.

Cells lacking functional Rad18 are able to initiate a DNA damage checkpoint arrest. HA-Chk1 (Chk1) is phosphorylated (Chk1P) in response to irradiation in rad18-dn and rad18-so cells in a manner similar to that seen with the controls (Chk1). Western blot analysis with the 12CA5 antibody was used to detect the forms of HA-Chk1, including a slower-migrating form that is phosphorylated in response to DNA damage. Chk1 activity is also induced in response to DNA damage. Chk1-HA was immunoprecipitated from vector or rad18-dn cell extracts and assayed for protein kinase activity (A [bottom panel]). Chk1-HA increased in response to DNA damage in rad18-so cells in a manner similar to wild-type cells (B [bottom panel]). The data shown represent the means of three independent experiments, with error bars showing the standard errors. In all cases, time zero corresponds to unirradiated cells. Wild-type cells grown in thiamine for 12.5 or 18 h did not differ significantly in their responses to UV-C treatment.

FIG. 7.

FIG. 7.

Nse1 is an essential non-SMC component of the Smc5/6 complex. (A) The nse1 gene contains one intron (indicated as a grey-shaded panel [left panel]). This was confirmed by comparing the results of PCR analysis using a cDNA template and primers 1 and 2 (lane 1) or 1 and 3 (lane 2) to the results obtained with the same primer combinations and a genomic DNA template (lanes 3 and 4) (right panel). (B) Alignment of human, mouse, and yeast Nse1 proteins. Nse1 is conserved across species, with the human, mouse, and S. pombe Nse1 proteins showing several regions of high homology, while S. cerevisiae (CEREV) Nse1 contains several inserts that are not homologous with its eukaryotic counterparts. (C) S. pombe cell extracts were prepared from wild-type cells or from cells in which Rad18 was expressed from the endogenous locus as an N-terminal triple-HA-tagged protein. Cells carrying either a vector or pREP42-Myc-Nse1 were assayed by IP and probed with the indicated antibodies. HA-Rad18 was detectable in extracts immunoprecipitated with the anti-Myc antibody, 9E10 (lane 4, bottom panel). IB, immunoblot. (D) Human Smc6 coimmunoprecipitates with Nse1. HEK293T cells were transfected with either empty vector or HA-tagged human Nse1. Where indicated, cells were treated with nocodazole to arrest cells in mitosis, a point at which Smc6 is no longer associated with chromatin and is more readily extracted. Cell extracts were transferred to nitrocellulose, the top portion of the filter was assayed (using anti-Smc6 polyclonal antibodies) for Smc6 expression in whole-cell extracts, and the lower portion was probed with anti-HA (12CA5) monoclonal antibodies to detect Nse1-HA (left panels). Anti-HA immunoprecipitates were similarly analyzed by Western blotting (right panels).

FIG.8.

FIG.8.

The Smc5/6 complex is essential for cell cycle arrest following DNA damage. (A) To assess the kinetics of aberrant mitosis in _nse1_Δ cells, spores from an nse1+/nse1::ura4 heterozygous diploid were germinated until the appearance of polar growth (10 h) at 30°C in medium lacking uracil to select for nse1::ura4. rad18::ura4 and rad18+ nse1+ spores were used as controls. DNA-damaging agents (MMS and 4-NQO) were then added, or cultures were left untreated (control); the results were monitored for a further 8 h. (B) Samples were taken, fixed, and stained with DAPI to assess the proportion and type of abnormal mitoses. nse1::ura4 cells manifested abnormalities 1 to 2 cell cycles later than rad18::ura4 cells. cont., control. (C) Examples of untreated (control) and 4-NQO- and MMS-treated nse1::ura4 and nse1+ cells after 8 h.

Similar articles

Cited by

References

    1. al-Khodairy, F., and A. M. Carr. 1992. DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J. 11:1343-1350. - PMC - PubMed
    1. Aono, N., T. Sutani, T. Tomonaga, S. Mochida, and M. Yanagida. 2002. Cnd2 has dual roles in mitotic condensation and interphase. Nature 417:197-202. - PubMed
    1. Barr, S. M., C. G. Leung, E. E. Chang, and K. A. Cimprich. 2003. ATR kinase activity regulates the intranuclear translocation of ATR and RPA following ionizing radiation. Curr. Biol. 13:1047-1051. - PubMed
    1. Basi, G., E. Schmid, and K. Maundrell. 1993. TATA box mutations in the Schizosaccharomyces pombe nmt1 promoter affect transcription efficiency but not the transcription start point or thiamine repressibility. Gene 123:131-136. - PubMed
    1. Bhat, M. A., A. V. Philp, D. M. Glover, and H. J. Bellen. 1996. Chromatid segregation at anaphase requires the barren product, a novel chromosome-associated protein that interacts with topoisomerase II. Cell 87:1103-1114. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources