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. 2007 Oct;27(19):6948-61.
doi: 10.1128/MCB.00774-07. Epub 2007 Jul 30.

The Ulp2 SUMO protease is required for cell division following termination of the DNA damage checkpoint

David C Schwartz  1 , Rachael Felberbaum, Mark Hochstrasser
Affiliations

The Ulp2 SUMO protease is required for cell division following termination of the DNA damage checkpoint

David C Schwartz et al. Mol Cell Biol. 2007 Oct.

Abstract

Eukaryotic genome integrity is maintained via a DNA damage checkpoint that recognizes DNA damage and halts the cell cycle at metaphase, allowing time for repair. Checkpoint signaling is eventually terminated so that the cell cycle can resume. How cells restart cell division following checkpoint termination is poorly understood. Here we show that the SUMO protease Ulp2 is required for resumption of cell division following DNA damage-induced arrest in Saccharomyces cerevisiae, although it is not required for DNA double-strand break repair. The Rad53 branch of the checkpoint pathway generates a signal countered by Ulp2 activity following DNA damage. Interestingly, unlike previously characterized adaptation mutants, ulp2Delta mutants do not show persistent Rad53 phosphorylation following DNA damage, suggesting checkpoint signaling has been terminated and no longer asserts an arrest in these cells. Using Cdc14 localization as a cell cycle indicator, we show that nearly half of cells lacking Ulp2 can escape a checkpoint-induced metaphase arrest despite their inability to divide again. Moreover, half of permanently arrested ulp2Delta cells show evidence of an aberrant mitotic spindle, suggesting that Ulp2 is required for proper spindle dynamics during cell cycle resumption following a DNA damage-induced cell cycle arrest.

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Figures

FIG. 1.
FIG. 1.
Ulp2 is required for cell division following DNA damage. (A) Outline of the DNA damage checkpoint pathway. (B) The ulp2Δ mutant is sensitive to DNA-damaging agents. (C) The ulp2Δ mutant is sensitive to very low levels of HU. (D) The ulp2Δ mutant is sensitive to transient exposure to high HU levels. (E) ulp2Δ cells arrest division following DNA damage from a single HO endonuclease-derived DSB. Fivefold serial dilutions of yeast cultures were spotted onto plates for panels B to D. Cells were photographed using Nomarski optics, and all plates were grown at 24°C for 4 days.
FIG. 2.
FIG. 2.
Ulp2 is not required for DNA repair by SSA. (A) Diagram of SSA repair assay. Galactose-induced HO cuts at a cleavage site inserted in leu2. 5′-to-3′ resection eventually reaches homologous DNA 30 kb away (depicted as a shaded rectangle). Repair occurs in a Rad52-dependent manner and results in a deletion of the intervening DNA. (B) ulp2Δ cells can repair an HO-induced DSB by SSA, but rad52Δ cells cannot. Control cells were grown overnight in YPD. Cells induced with galactose were first grown overnight in yeast extract-peptone (YEP) raffinose and then transferred to YEP galactose medium for a second night. All samples were grown at 24°C. Genomic DNA was used as a template for PCR analysis. P1, P2, and P3 are primers used for PCR (annealing sites are depicted in panel A). (C) ulp2Δ cells have a growth defect in response to a repairable DSB. Tenfold serial dilutions of yeast cultures were spotted onto glucose or galactose plates and grown for 4 days at 24°C.
FIG. 3.
FIG. 3.
Ulp2 is required for cell division following induction of a nonrepairable DSB. Cells were grown in liquid medium containing galactose for 1 h to induce the HO endonuclease and then micromanipulated onto plates containing galactose. Black bars represent the percentage of cells in each microcolony size category after 24 h with DNA damage, and the gray bars represent the percentage of cells in each category at the end of the experiment (72 h). The percentage arrested after 24 h is noted at left, and the percentage noted at right is the percentage of microcolonies containing >3 cells after 72 h. (A) Following transient cell cycle arrest, WT cells adapt to DNA damage and undergo multiple cell divisions (see inset; 72 h). The ulp2Δ cells permanently arrest as large-budded cells following damage. (B) rad51Δ and rad52Δ cells adapt to and divide after DNA damage. (C) mec1Δ and mec1Δ ulp2Δ cells show defective arrest following DNA damage. Note that in the double mutant, which grows very poorly, a large fraction likely fails to divide further as a result of this general growth defect.
FIG. 4.
FIG. 4.
ulp2Δ cells without DNA damage were able to form viable colonies. Bars for panels A and B are as in Fig. 3. (A) ulp2Δ strains lacking an HO cut site. (B) ulp2Δ cells with an HO cut site that were not induced for HO expression with galactose. (C) ulp2Δ cells treated for 8 h with either HU (0.2 M) or water can form microcolonies.
FIG. 5.
FIG. 5.
Ulp2 is required downstream of the Rad53 branch of the checkpoint pathway for cell division following nonrepairable DNA damage. Bars are as in Fig. 3. (A) Both rad53Δ and rad53Δ ulp2Δ cells show impaired arrest and undergo multiple divisions in the presence of DNA damage. (B) The chk1Δ mutant shows impaired arrest, but the chk1Δ ulp2Δ mutant is permanently arrested following DNA damage. (C) The dun1Δ and dun1Δ ulp2Δ mutants continue to divide in the presence of DNA damage. (D) The pds1Δ and pds1Δ ulp2Δ mutants show impaired arrest following DNA damage.
FIG. 6.
FIG. 6.
SUMO protease activity of Ulp2 is required for cell division following DNA damage, but only when sumoylation is unimpaired. Bars are as in Fig. 3. (A) A WT copy of ULP2 on a plasmid rescues ulp2Δ permanent arrest, whereas a plasmid-borne ulp2-H531A catalytically inactive allele fails to do so. (B) Sumoylation is not required for DNA damage checkpoint arrest or the resumption of cell division following arrest. Cells were shifted to 37°C, the restrictive temperature for ubc9-1 cells, for 4 h. Galactose was added to the medium to induce HO, and cells were incubated at 37°C for 1 h longer and then manipulated onto galactose plates and incubated at 24°C for 4 days. Both WT and ubc9-1 strains arrest following DNA damage and subsequently adapt and divide. (C) The siz1Δ siz2Δ strain is able to arrest following damage and then adapt and divide (experiment done at 24°C). (D) The permanent arrest of ulp2Δ cells following DNA damage is suppressed by ubc9-1.
FIG. 7.
FIG. 7.
Rad53 is dephosphorylated in ulp2Δ cells as in WT cells, indicating termination of DNA checkpoint signaling. (A) WT and ulp2Δ strains show similar Rad53 phosphorylation and dephosphorylation kinetics following nonrepairable DNA damage. Cells were incubated with galactose for 4 h to induce DNA damage and then placed into glucose-containing medium (times shown are from the addition of galactose). (B) Ptc2 overexpression does not suppress ulp2Δ cell cycle arrest, in contrast to the case with srs2Δ cells. Bars are as in Fig. 3.
FIG. 8.
FIG. 8.
ulp2Δ cells can escape DNA damage-induced metaphase arrest. Cells were grown at 24°C in liquid medium containing galactose for 1 h to induce HO and then micromanipulated onto plates containing galactose. Cdc14-GFP was observed by fluorescence microscopy after either 1 or 2 days (d) (1 day = 20 to 31 h; 2 days = 44.5 to 49.5 h). (A) Representative arrested WT, ulp2Δ, or srs2Δ cells 1 or 2 days after galactose induction. WT cells divide after 1 day. Arrows indicate Cdc14-GFP. (B) Forty-two percent of ulp2Δ cells (and 23% of srs2Δ cells) show Cdc14 in both cells by 2 days. Pictures were scored in groups of 40, and 120 cells of each genotype were counted. Error bars represent standard deviations.
FIG. 9.
FIG. 9.
Aberrant spindles in ulp2Δ cells following DNA damage-induced arrest. Cells were grown as described in the legend to Fig. 8. Tub1-GFP was observed by fluorescence microscopy after either 1 or 2 days(d) (1 day = 19.5 to 28.5 h; 2 days = 48 to 52 h). (A) Pictures of representative WT, ulp2Δ, or srs2Δ cells 1 or 2 days after galactose induction. (B) Fifty percent of ulp2Δ cells (and 18% of srs2Δ cells) show noncontiguous Tub1-GFP fluorescence at 2 days. Pictures were scored in groups of 40, and at least 120 cells of each genotype were counted. Between 1 and 3 focal planes per cell were examined to ensure the entire GFP signal was scored. Error bars represent standard deviations. Cells were scored as being in metaphase/early anaphase if the spindle was short and either at the bud neck or slightly protruded into the bud, in telophase if the spindle stretched from one end of the mother to the opposite end of the bud, or as having an aberrant spindle if the spindle appeared as disconnected segments.
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