hMMS21 is sumoylated in vitro. (A) hMMS21 is sumoylated in vitro. myc-hMMS21 was in vitro translated in the presence of [³⁵S]methionine and incubated with SUMO reaction mixtures containing the indicated components and analyzed by SDS-PAGE followed by autoradiography. (B) Sumoylation of hMMS21 in vitro requires an intact SP-RING domain. SUMO reactions were performed as in panel A using either wild type (WT) or the indicated mutants of hMMS21. The percentage of hMMS21 sumoylated was quantitated and is shown below the appropriate lanes. (C) hMMS21 is conjugated to SUMO1 in vitro. In vitro SUMO reactions were performed using GST-hMMS21 and the indicated components. GST-hMMS21 was pulled down using glutathione-agarose beads followed by SDS-PAGE and immunoblotting with anti-GST. (D) The reaction mixtures described in panel C were immunoblotted with anti-SUMO1. WB, Western blot.

hMMS21 is a SUMO ligase that stimulates sumoylation of hSMC6 and TRAX. (A) Recombinant GST-hMMS21 stimulates the sumoylation of TRAX, but not ETV1, in vitro. ETV1 and TRAX were in vitro translated in the presence of [³⁵S]methionine and subjected to SUMO reactions in the presence of GST or GST-hMMS21. The reactions were analyzed by SDS-PAGE followed by autoradiography. The fold increase of the monosumoylated TRAX in the presence of GST-hMMS21 is indicated. (B) Sumoylation of hSMC6 and TRAX, but not ETV1, is enhanced by hMMS21 in HeLa cells. myc-TRAX, myc-hSMC6, or myc-ETV1 was expressed with GFP-SUMO1 wild type (WT) or ΔGG in the presence or absence of wild-type or C215A mutant GFP-hMMS21 in HeLa cells. The whole-cell lysates were blotted with anti-myc. (C) hMMS21 stimulates SUMO1 conjugation to hSMC6 in HeLa cells. HeLa cells were transfected as described in panel B, and whole-cell lysates were either immunoblotted with anti-myc (left panel) or immunoprecipitated with anti-myc followed by immunoblotting with anti-SUMO1 (right panel). IP, immunoprecipitation; WB, Western blot.

ATM/ATR are hyperactivated in hMMS21-RNAi cells after DNA damage. (A) HeLa cells were transfected with either mock or hMMS21 siRNA for 48 h and were either untreated or treated with 0.015% MMS for 4 h. Cells were fixed and stained with Hoechst 33258 (blue) or a phosphospecific antibody against ATM/ATR phosphorylation sites on its substrates (green). Bar, 5 μm. (B) Quantitation of the fluorescence intensities of the ATM/ATR phosphosubstrate staining in panel A. Results from three separate experiments are averaged with the standard deviation indicated. (C) Cells treated as in panel A were stained with a phospho-T68 CHK2 antibody (green). Bar, 5 μm. (D) Histogram of cells in panel C with the indicated number of phospho-CHK2 foci.

hMMS21 is a subunit of the hSMC5/6 complex. (A) Sequence identities among MMS21 proteins of H. sapiens, S. cerevisiae, and S. pombe. The identities of the full-length hMMS21, the N-terminal domain, and the C-terminal SP-RING domain (indicated by dashed lines) are shown. (B) Sequence alignment of the SP-RING domains of PIAS1 and the MMS21 proteins from human (Hs), S. cerevisiae (Sc), and S. pombe (Sp). The conserved residues are shaded. The putative Zn²⁺-coordinating cysteines and histidines are indicated by asterisks. Alignment was performed using ClustalW. (C) hMMS21 and hNSE1 interact with hSMC5. The pCS2-myc-hSMC5 plasmid was transfected into HeLa cells with or without cotransfection of plasmids encoding HA-tagged hMMS21 or hNSE1. Lysates of these cells were immunoprecipitated with anti-HA and blotted with anti-myc. The whole-cell lysates (WCL) were also blotted with anti-myc. IP, immunoprecipitation; WB, Western blot. (D) The hSMC5 antibody recognizes endogenous hSMC5 in HeLa cells. HeLa cells were transfected with mock siRNA or hSMC5 siRNA in the presence or absence of pCS2-myc-hSMC5. Lysates of these cells were blotted with anti-hSMC5 antibody. The asterisk indicates a nonspecific cross-reacting band that serves as a loading control. (E) The hMMS21 antibody recognizes endogenous hMMS21 in HeLa cells. HeLa cells were transfected with mock siRNA or hMMS21 siRNA for 48 h. Lysates of these cells were blotted with anti-hMMS21. The asterisk indicates a nonspecific cross-reacting band that serves as a loading control. (F) Endogenous hMMS21 interacts with hSMC5 in HeLa cells. HeLa cell lysates were immunoprecipitated with anti-GST, anti-BUBR1, or anti-hSMC5 and blotted with anti-hMMS21. Immunoglobulin G (IgG) heavy chain was used as a loading control. IP, immunoprecipitation; WCL, whole-cell lysate.

hMMS21 is sumoylated in HeLa cells. (A) hMMS21 is sumoylated in vivo. myc-hMMS21 was expressed in HeLa cells with or without untagged (UT) or GFP-tagged (GFP) SUMO1 or the SUMO isopeptidase SENP2. Whole-cell lysates were blotted with anti-myc. (B) Sumoylation of hMMS21 in vivo requires an intact RING domain. The wild type (WT) or the C215A mutant of myc-hMMS21 was expressed in HeLa cells in the presence of WT or ΔGG mutant GFP-SUMO1 and with or without SENP2. Whole-cell lysates were blotted with anti-myc. (C) hMMS21 is conjugated to SUMO1 in HeLa cells. HeLa cells were transfected as described in panel B with either the wild type (WT) or the C215A mutant of myc-hMMS21 in the presence of WT or ΔGG mutant GFP-SUMO1. Whole-cell lysates were blotted with anti-myc (left panel) or immunoprecipitated with anti-myc followed by immunoblotting with anti-SUMO1 (right panel). IP, immunoprecipitation; WB, Western blot.

The SUMO ligase activity of hMMS21 is required to block DNA damage-induced apoptosis. (A) hMMS21-RNAi cells are more sensitive to MMS. HeLa cells were transfected with mock or hMMS21 siRNA for 48 h and then either untreated or treated with 0.015% MMS for 6 h. The percentage of dead cells (trypan blue positive) is plotted. Results from three separate experiments are averaged with the standard deviation indicated. (B) Time course of cell death after MMS and etoposide treatment. Control (squares) or hMMS21-RNAi (circles) cells were treated with 0.015% MMS (solid lines) or 40 μg/ml etoposide (dashed lines) for the indicated times, and the percentage of dead cells was determined by trypan blue exclusion. (C) The wild-type (WT) hMMS21, but not its ligase-inactive C215A mutant, rescues the sensitivity of hMMS21-RNAi cells to MMS. Cells were transfected with mock or hMMS21 siRNA for 24 h and then transfected with either empty vector or plasmids encoding the wild type or the C215A mutant of myc-hMMS21. After 24 h, cells were treated with 0.015% MMS for 6 h. The percentage of dead cells (trypan blue positive) is plotted. Results from three separate experiments are averaged with the standard deviation indicated. (D) Whole-cell lysates of cells from panel C were blotted with antibodies against cleaved PARP or anti-hMMS21. The asterisk indicates a nonspecific cross-reacting band that serves as a loading control.

hMMS21 is required for efficient repair of damaged DNA. (A) HeLa cells were transfected with mock or hMMS21 siRNA and treated with 0.015% MMS for 0 h (−MMS), with MMS for 1 h followed by a 3-h recovery period without MMS (+MMS), with MMS for 1 h with 3 h of recovery in the presence of the pan-caspase inhibitor zVAD-fmk (+MMS + zVAD), or with MMS for 1 h without recovery (+MMS No Recovery). Cells were collected and analyzed for unrepaired DNA lesions by the comet assay. SYBR green staining of DNA shows comet tails migrating out of the nucleus. Bar, 10 μm. (B) Quantitation of fluorescence intensities of cells in panel A to measure the percentage of DNA migrating out of the nucleus into tails. Results from three separate experiments are averaged with the standard deviation indicated.