A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization.

Through a genetic screen using myosin-like protein strains mlp1Delta mlp2Delta and biochemical purification, we identified a complex of eight proteins, each required for growth and DNA repair in Saccharomyces cerevisiae. Among the subunits are Mms21 that contains a putative Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain characteristic of small ubiquitin-like modifier (SUMO) ligases, two structural-maintenance-of-chromosome (Smc) proteins, Smc5 and Smc6, and a protein that contains an ubiquitin ligase signature domain. We show that these proteins colocalized to several distinct nuclear foci. Biochemical and genetic data demonstrated that Mms21 indeed functions as a SUMO ligase and that this activity requires the Siz/PIAS (protein inhibitor of activated signal transducer and activator of transcription) RING domain. The substrates for this SUMO ligase include a subunit of the octameric complex, Smc5, and the DNA repair protein Yku70. We further show that the abolition of the SUMO E3 activity of Mms21 leads to such disparate phenotypes as DNA damage sensitivity, defects in nucleolar integrity and telomere clustering, silencing, and length regulation. We propose that Mms21 sumoylates proteins involved in these diverse processes and that the other members of the complex, particularly Smc5/6, facilitate proper substrate sumoylation by localizing Mms21 to specific chromosomal regions.

[1]  K. Nasmyth,et al.  Building and breaking bridges between sister chromatids. , 2003, BioEssays : news and reviews in molecular, cellular and developmental biology.

[2]  G. Blobel,et al.  Cell Cycle–Regulated Attachment of the Ubiquitin-Related Protein Sumo to the Yeast Septins , 1999, The Journal of cell biology.

[3]  M. Hochstrasser,et al.  The Yeast ULP2 (SMT4) Gene Encodes a Novel Protease Specific for the Ubiquitin-Like Smt3 Protein , 2000, Molecular and Cellular Biology.

[4]  Kenji Kohno,et al.  Identification of a Novel Non-structural Maintenance of Chromosomes (SMC) Component of the SMC5-SMC6 Complex Involved in DNA Repair* , 2002, The Journal of Biological Chemistry.

[5]  T. Hirano The ABCs of SMC proteins: two-armed ATPases for chromosome condensation, cohesion, and repair. , 2002, Genes & development.

[6]  V. G. Panse,et al.  Unconventional tethering of Ulp1 to the transport channel of the nuclear pore complex by karyopherins , 2003, Nature Cell Biology.

[7]  M. Mann,et al.  Mlp2p, A Component of Nuclear Pore Attached Intranuclear Filaments, Associates with Nic96p* , 2000, The Journal of Biological Chemistry.

[8]  Erica S. Johnson,et al.  Protein modification by SUMO. , 2004, Annual review of biochemistry.

[9]  K. Runge,et al.  Two paralogs involved in transcriptional silencing that antagonistically control yeast life span , 2000, Current Biology.

[10]  J. Yates,et al.  Novel Essential DNA Repair Proteins Nse1 and Nse2 Are Subunits of the Fission Yeast Smc5-Smc6 Complex* , 2003, Journal of Biological Chemistry.

[11]  H. Scherthan,et al.  The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae , 1996, The Journal of cell biology.

[12]  M. Hochstrasser,et al.  A new protease required for cell-cycle progression in yeast , 1999, Nature.

[13]  L. Prakash,et al.  Hyper-recombination and mutator effects of the mms9-1, mms13-1, and mms21-1 mutations in Saccharomyces cerevisiae , 1981, Current Genetics.

[14]  H. Yasuda,et al.  Yeast Ull1/Siz1 Is a Novel SUMO1/Smt3 Ligase for Septin Components and Functions as an Adaptor between Conjugating Enzyme and Substrates* , 2001, The Journal of Biological Chemistry.

[15]  R. Jessberger The many functions of smc proteins in chromosome dynamics , 2002, Nature Reviews Molecular Cell Biology.

[16]  M. Resnick,et al.  Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae. , 2000, Mutation research.

[17]  S. Jentsch,et al.  Ubiquitin and proteasomes: Sumo, ubiquitin's mysterious cousin , 2001, Nature Reviews Molecular Cell Biology.

[18]  G. Gill,et al.  SUMO and ubiquitin in the nucleus: different functions, similar mechanisms? , 2004, Genes & development.

[19]  A. Lehmann,et al.  A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein , 2000, The EMBO journal.

[20]  M. Hochstrasser,et al.  The Ulp1 SUMO isopeptidase , 2003, The Journal of cell biology.

[21]  B. Meyer,et al.  Condensin and cohesin: more than chromosome compactor and glue , 2003, Nature Reviews Genetics.

[22]  A. Tomkinson,et al.  Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. , 2001, Molecular cell.

[23]  M. Hochstrasser SP-RING for SUMO New Functions Bloom for a Ubiquitin-like Protein , 2001, Cell.

[24]  R. Rothstein,et al.  Rad52 forms DNA repair and recombination centers during S phase , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[25]  Erica S. Johnson,et al.  An E3-like Factor that Promotes SUMO Conjugation to the Yeast Septins , 2001, Cell.

[26]  M. Tatham,et al.  Polymeric Chains of SUMO-2 and SUMO-3 Are Conjugated to Protein Substrates by SAE1/SAE2 and Ubc9* , 2001, The Journal of Biological Chemistry.

[27]  C. Slaughter,et al.  Identification of a Multifunctional Binding Site on Ubc9p Required for Smt3p Conjugation* , 2002, The Journal of Biological Chemistry.

[28]  F. Z. Watts,et al.  The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair , 1995, Molecular and cellular biology.

[29]  A. Ui,et al.  SMC6 is required for MMS-induced interchromosomal and sister chromatid recombinations in Saccharomyces cerevisiae. , 2004, DNA repair.

[30]  D. Botstein,et al.  A new yeast gene with a myosin-like heptad repeat structure , 1993, Molecular and General Genetics MGG.

[31]  T. Petes,et al.  Interactions of TLC1 (Which Encodes the RNA Subunit of Telomerase), TEL1, and MEC1 in Regulating Telomere Length in the Yeast Saccharomyces cerevisiae , 1999, Molecular and Cellular Biology.

[32]  G. Blobel,et al.  Mlp-dependent anchorage and stabilization of a desumoylating enzyme is required to prevent clonal lethality , 2004, The Journal of cell biology.

[33]  Brian E Snydsman,et al.  Assigning function to yeast proteins by integration of technologies. , 2003, Molecular cell.

[34]  A. Dejean,et al.  Nuclear and unclear functions of SUMO , 2003, Nature Reviews Molecular Cell Biology.

[35]  F. Melchior,et al.  SUMO--nonclassical ubiquitin. , 2000, Annual review of cell and developmental biology.

[36]  J. Fuchs,et al.  Centromere clustering is a major determinant of yeast interphase nuclear organization. , 2000, Journal of cell science.

[37]  E. Kremmer,et al.  A Short C-terminal Domain of Yku70p Is Essential for Telomere Maintenance* , 2000, The Journal of Biological Chemistry.

[38]  Michael P. Rout,et al.  Proteins Connecting the Nuclear Pore Complex with the Nuclear Interior , 1999, The Journal of cell biology.

[39]  P B Moens,et al.  Characterization of a novel human SMC heterodimer homologous to the Schizosaccharomyces pombe Rad18/Spr18 complex. , 2001, Molecular biology of the cell.

[40]  Satya Prakash,et al.  Increased spontaneous mitotic segregation in MMS-sensitive mutants of Saccharomyces cerevisiae. , 1977, Genetics.

[41]  R. Dohmen,et al.  SUMO conjugation and deconjugation , 2000, Molecular and General Genetics MGG.

[42]  Jean-Christophe Olivo-Marin,et al.  Nuclear pore complexes in the organization of silent telomeric chromatin , 2000, Nature.

[43]  Boris Pfander,et al.  RAD6-dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO , 2002, Nature.

[44]  O. Gadal,et al.  Nuclear Retention of Unspliced mRNAs in Yeast Is Mediated by Perinuclear Mlp1 , 2004, Cell.