Genotoxic stress sensitivity of strains having point mutations in conserved residues of SPL-RING domain of Mms21. A, multiple sequence alignment showing conserved residues (depicted in bold) of S. cerevisiae Mms21 (His-202 and Cys-221) that were mutated to alanine in this study. Other known functionally important residues of S. cerevisiae and S. pombe Mms21/Nse2 proteins are underlined. “*” denotes identical residues (100% identity), “:” denotes conserved substitutions, and “.” denotes semiconserved substitutions. The color denotes regions having 100% identity (red), 80–90% identity (magenta), and 50–70% identity (blue) among the Mms21-related proteins shown. The sequences included are H. sapiens (Hs), E. caballus (Ec), S. salar (Ss), A. thaliana (At), O. sativa Indica group (Os), A. gambiae (Ag), S. pombe (Sp), S. cerevisiae (Sc), C. elegans (Ce), and P. vivax (Pv). B, HU and MMS sensitivity of mutants having C221A and H202A substitutions. Haploid strains having the mms21Δsl mutation (SLY792) and yeast centromeric plasmid pRS314 (no insert), pRR51 (expressing MMS21), pRR55 (mms21-C221A), or pRR56 (mms21-H202A) were grown to midlog phase, serially diluted, spotted onto an SC-Trp (Trp omission) plate and SC-Trp plates containing 100 mm HU or 0.02% MMS, and incubated at 23 °C. Images were recorded after 7 days in the case of SC-Trp and 100 mm HU plates and after 9 days in the case of 0.02% MMS-containing plates. C, slow growth of unchallenged strains having the mms21-C221A and mms21-H202A point mutations. A haploid strain (SLY782) having the mms21::KanMX6 null allele complemented by pRR50 having MMS21 on a centromeric URA3-bearing plasmid was transformed with yeast centromeric plasmid pRS314 (no insert; indicated by −), pRR51 (expressing MMS21; indicated by WT), pRR55 (mms21-C221A; indicated by C-A), or pRR56 (mms21-H202A; indicated by H-A) and subjected to counterselection on SC-Trp 5-FOA plates. Top panel, growth on counterselective medium (3 days) following one round of incubation on 5-FOA. Bottom panel, growth of strains (which had lost pRR50 after plasmid shuffling) having WT (SLY1620), mms21-C221A (C-A) (SLY1621) point mutation, or mms21-H202A (H-A) (SLY1622) point mutation on YPD (2.5 days), 100 mm HU (9 days), or SC-Trp (3 days) plates. D, Western blot analysis of lysates from cells expressing HA epitope-tagged mms21-H202A, mms21-C221A, and MMS21 proteins using anti-HA antibody. U, untagged lysate. The arrow indicates the position of 3HA-tagged Mms21 proteins. The bottom panel is a Ponceau S-stained image of the same blot showing equivalent loading of samples.

A, genetic interactions of mms21Δsl with mutants defective in known caffeine targets. WT (VP106), mec1 (also having the sml1 mutation), tel1 (SLY1353), mec1 mms21Δsl (also having the sml1 mutation; SLY1412), and tel1 mms21Δsl (SLY1416) strains were grown to midlog phase, serially diluted, and spotted on YPD plates (top panels) and further analyzed for UV radiation (500 μJ/cm²) or MMS (0.0025%) sensitivity (middle panels). The bottom panels show WT (Jka-3da), tor2 (JK350-18a), tor1 tor2 (JK350-21a), mms21Δsl (SLY1552), and tor1 tor2 mms21Δsl (SLY1549) strains that were grown to midlog phase, serially diluted, spotted on YPD plates, and incubated for 3 or 4 days at 23 °C. B, genetic interaction of mms21-C221A and mms21-H202A point mutants with mec1. A haploid strain having the mms21Δsl and mec1 mutations (SLY1412) was transformed with the centromeric plasmid pRS314 (no insert; top left), pRR51(expressing MMS21; top right), pRR55(mms21-C221A; bottom left), or pRR56 (mms21-H202A; bottom right); plated on SC-Trp plates; and incubated at 23 °C. Images were recorded 3 and 4.5 days after plating. Representative data from three independent transformation experiments are shown.

Genetic interactions of mms21Δsl with mutants defective in replication stress and DNA damage checkpoint pathways. A, the mms21Δsl mutant cells are HU-sensitive. WT (YPH499) and mms21Δsl (SLY792) strains were grown to midlog phase, serially diluted, spotted onto a YAPD plate containing 150 mm HU, and incubated at 23 °C for 5 days. B, genetic interaction of mms21Δsl with checkpoint-defective mutants mrc1 and rad9. WT (Y1134), mrc1 (Y1127), mms21Δsl (SLY1523), mrc1 mms21Δsl (SLY1516), rad9 (Y2297), and rad9 mms21Δsl (SLY1526) strains were grown to midlog phase, serially diluted, spotted on adenine supplemented YPD plates or YAPD plates having the indicated concentrations of MMS and HU, and incubated for 3 days. C, genetic interaction of mms21Δsl with DNA damage checkpoint-defective mutants rad9 and rad24. WT (Y1134), mms21Δsl (SLY1523), rad9 (Y2297), rad9 mms21Δsl (SLY1526), rad24 (SLY1534), and rad24 mms21Δsl (SLY1543) strains were grown to midlog phase and analyzed for MMS and HU sensitivity.

Activation of mitotic checkpoint pathways in SUMO ligase-deficient cells. A, analysis of the activity of the replication stress checkpoint pathway. Wild-type (MMS21) or mms21Δsl cells having HA-tagged Mrc1 were subjected to HU-induced replication stress for varying time periods up to 3.5 h (left panels), and total lysates were analyzed by SDS-PAGE followed by Western blotting using anti-HA antibody (left panels). Modification of Mrc1 was observed in wild-type (top left panel) as well as mutant (bottom left panel) cell extracts. Cells were released from HU-induced arrest into YAPD medium lacking HU for varying time periods and analyzed for persistence of modification of Mrc1 (right panels), which was preferentially observed in the case of mutant cells. Mrc1 indicates the location of unmodified form; the arrow indicates the location of replication stress-induced modified forms. Similarly, wild-type (MMS21) or mms21Δsl cells having HA-tagged Rad53 were analyzed to detect the formation of HU-induced modified forms of Rad53 (position indicated by arrows). A, asynchronous cells prior to addition of HU. The lower panels are the corresponding regions from the Ponceau S-stained Western blots. B, activation of the spindle assembly checkpoint pathway in replication-stressed cells that were released into nocodazole (30 μg/ml)-containing medium for varying time intervals. Total lysates from wild-type (left) or mms21Δsl (right) cells were analyzed by SDS-PAGE followed by Western blotting using anti-Mad1 antibody (top panels). A, asynchronous cells prior to addition of HU; HU, cells treated with 150 mm HU for 3.5 h; the arrow indicates the position of the modified form of Mad1 that was observed after treatment with nocodazole. The lower panels are the corresponding regions from the Ponceau S-stained Western blots. Histograms show the results of FACS analysis, confirming mitotic arrest in nocodazole-treated wild-type (MMS21; left) and mms21Δsl (right) cell populations that were treated with HU for 3.5 h and released into nocodazole-containing medium (from bottom to top: asynchronous, 150 mm HU for 3.5 h, and released into nocodazole (30 μg/ml)-containing medium for 0.5, 1, 1.5, 2, 3, and 4 h).

Characterization of growth properties of SUMO ligase-defective mms21Δsl mutant. A, sensitivity of mms21Δsl cells to DNA damage induction. A schematic showing the strategy for creation of mms21Δsl::KanMX6 mutant is shown at the top. To confirm the sensitivity of the nse2Δsl mutant cells to extrinsic genotoxic stress, WT (YPH499) and mms21Δsl (SLY792) strains were grown to midlog phase, serially diluted (10-fold), spotted onto YPD containing MMS (0.02%) or bleomycin (5 μg/ml) or subjected to UV irradiation (4000 μJ/cm²), and incubated for 3–4 days at 23 °C. B, slow growth of unchallenged mms21Δsl cells. WT (YPH499) and mms21Δsl (SLY792) strains were grown to midlog phase, serially diluted, spotted on a YPD plate, and incubated for 3 days. C, the graph depicts the slow growth of mms21Δsl mutant cells (circles) relative to wild-type cells (squares) in liquid medium. D, accumulation of checkpoint signals in unchallenged mms21Δsl mutant cells. Whole cell extracts from midlog phase asynchronous cultures of MRC1-3HA (SLY861), Mrc1-3HA/mms21Δsl (SLY872), Rad53-3HA (SLY1100), Rad53-3HA/mms21Δsl (SLY1104), Rad9-3HA (SLY1095), and Rad9-3HA/mms21Δsl (SLY1098) strains were resolved by SDS-PAGE and analyzed by immunoblotting using anti-HA antibody. The arrows indicate the locations of modified forms of the activated checkpoint proteins Mrc1, Rad9, and Rad53. The lower panels are corresponding regions from the Ponceau S-stained Western blots.

Caffeine-sensitive checkpoint pathway signaling is important for survival of unchallenged mms21Δsl cells. A, caffeine-sensitive growth of mms21Δsl mutant cells. Wild type (SLY1351) and mms21Δsl (SLY1415) were grown to midlog phase, serially diluted, and spotted onto a YPD agar plate (top left) and YPD agar plates having the indicated concentrations of caffeine. B, caffeine overrides activation of Mrc1 in HU- and MMS-treated cells. HU (150 mm) or MMS (0.03%) was added to asynchronous cultures of strains Mrc1-3HA (SLY1351) and Mrc1-3HA/mms21Δsl (SLY1415) growing at 25 °C in YPD medium. After 2 h of incubation, caffeine (10 mg/ml) was added to one-half of the culture and further incubated for 1 h. Whole cell extracts were resolved by SDS-PAGE and analyzed by immunoblotting using anti-HA antibody. The arrow indicates the location of Mrc1, * indicates location of the replication stress-induced modified Mrc1, and the label B indicates an anti-HA cross-reactive background band present in these cell lysates. The label b indicates a blank lane, and u denotes a lane having lysate from the parental untagged strain. C, caffeine abrogates activation of Rad9 and Rad53 in unchallenged mms21Δsl cells. Rad53-3HA (SLY1100), Rad53-3HA/mms21Δsl (SLY1104), Rad9-3HA (SLY1095), and Rad9-3HA/mms21Δsl (SLY1098) strains were grown to midlog phase, caffeine (10 mg/ml) was added to one-half of the culture, and strains were incubated further for 1 h. Whole cell extracts were resolved by SDS-PAGE and analyzed by Western blotting using the anti-HA antibody. + indicates samples in which caffeine was added, − indicates that no caffeine was added, and * indicates the location of caffeine-sensitive modified bands of Rad9 (left) or Mrc1 (right).

Measurement of chromosome integrity in wild-type and mms21Δsl mutant cells. A, quantitative genetic assay for telomere marker loss and total YAC loss. The schematic shows the organization of the YACs used for the gross chromosomal rearrangement assay (adapted from Ref. 35). B, chromosome instability in mms21Δsl cells. Shown is the quantification of telomere marker loss (YAC integrity; top panels) and total YAC loss (YAC stability; bottom panels) plotted as percent loss on the y axis in WT (DH2004-1/101 and DH1003/102) and mms21Δsl (SLY1344 and SLY1348) cells that harbored the YACs PA3-I (left panels) and yWSS1572-1 (right panels). C, confirmation of telomere marker loss or chromosome breakage. Genomic DNA was prepared from randomly isolated Trp⁺ura⁻ mms21Δsl cells identified in B and subjected to PCR analysis (to amplify telomere-proximal markers ADE2 and URA3 or the centromere-proximal marker TRP1) to confirm chromosome breakage in samples scored as telomere marker loss events. M, molecular weight marker; WT, PCR analysis of DNA isolated from wild-type cells bearing the intact YAC.

Cell cycle arrest in SUMO ligase-deficient cells released from transient HU-induced replication stress. A, FACS profiles showing the distribution of cells having varying DNA content were determined for cells having the mms21Δsl mutation (SLY792) transformed with yeast centromeric plasmid pRR51 (expressing MMS21; top), pRS314 (no insert; middle; labeled as mms21Δsl), or pRR55 (mms21-C221A; bottom). In each case, the series of histograms represent (from bottom to top) untreated cycling cells having an A₆₀₀ of ∼0.2, cells treated with 150 mm hydroxyurea for 3 h, and cells that were released from HU-induced arrest for varying intervals (10 min, 30 min, 40 min, 60 min, 1 h 20 min, 1 h 40 min, 2 h, 2 h 20 min, 2 h 40 min, 3 h, 3.5 h, 4 h, 4.5 h, and 5 h). The arrows indicate the position of peaks corresponding to 1C and 2C DNA content. B, FACS analysis of strains having mms21::KanMX6 null allele complemented by centromeric plasmid expressing wild-type (MMS21, SLY1620; top panel) or SUMO ligase-defective mutant (mms21-C221A, SLY1621; bottom panel) Mms21 proteins. Samples were treated with HU as in A and released into YPD for varying intervals (20 min, 40 min, 1 h, 1 h 20 min, 1 h 40 min, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, and 10 h).