title: SUMO chain formation is required for response to replication arrest in S. pombe.
abstract: SUMO is a ubiquitin-like protein that is post-translationally attached to one or more lysine residues on target proteins. Despite having only 18% sequence identity with ubiquitin, SUMO contains the conserved betabetaalphabetabetaalphabeta fold present in ubiquitin. However, SUMO differs from ubiquitin in having an extended N-terminus. In S. pombe the N-terminus of SUMO/Pmt3 is significantly longer than those of SUMO in S. cerevisiae, human and Drosophila. Here we investigate the role of this N-terminal region. We have used two dimensional gel electrophoresis to demonstrate that S. pombe SUMO/Pmt3 is phosphorylated, and that this occurs on serine residues at the extreme N-terminus of the protein. Mutation of these residues (in pmt3-1) results in a dramatic reduction in both the levels of high Mr SUMO-containing species and of total SUMO/Pmt3, indicating that phosphorylation of SUMO/Pmt3 is required for its stability. Despite the significant reduction in high Mr SUMO-containing species, pmt3-1 cells do not display an aberrant cell morphology or sensitivity to genotoxins or stress. Additionally, we demonstrate that two lysine residues in the N-terminus of S. pombe SUMO/Pmt3 (K14 and K30) can act as acceptor sites for SUMO chain formation in vitro. Inability to form SUMO chains results in aberrant cell and nuclear morphologies, including stretched and fragmented chromatin. SUMO chain mutants are sensitive to the DNA synthesis inhibitor, hydroxyurea (HU), but not to other genotoxins, such as UV, MMS or CPT. This implies a role for SUMO chains in the response to replication arrest in S. pombe.
authors.affiliation: Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Skilton
authors.forename: Jenny C Y
authors.initials: JC
authors.lastname: Ho
authors.forename: Brenda
authors.initials: B
authors.lastname: Mercer
authors.forename: Emily
authors.initials: E
authors.lastname: Outwin
authors.forename: Felicity Z
authors.initials: FZ
authors.lastname: Watts
mesh_terms: D004249:DNA Damage; D015180:Electrophoresis, Gel, Two-Dimensional; D006918:Hydroxyurea / Q000494:pharmacology; D010766:Phosphorylation; D012568:Schizosaccharomyces / Q000166:cytology* / Q000187:drug effects; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000502:physiology*
chemical_list: D025841:Small Ubiquitin-Related Modifier Proteins; D006918:Hydroxyurea
references.citation: Mol Cell. 2008 Nov 7;32(3):301-5
references.pmid: 18995828
references.citation: J Biochem. 2003 Apr;133(4):415-22
references.pmid: 12761287
references.citation: Nucleic Acids Res. 2001 Oct 15;29(20):4179-86
references.pmid: 11600706
references.citation: Annu Rev Cell Dev Biol. 2006;22:159-80
references.pmid: 16753028
references.citation: Nature. 2003 Sep 11;425(6954):188-91
references.pmid: 12968183
references.citation: EMBO J. 2007 Sep 19;26(18):4102-12
references.pmid: 17762864
references.citation: EMBO J. 2007 Sep 19;26(18):4089-101
references.pmid: 17762865
references.citation: Trends Biochem Sci. 2001 May;26(5):332-3
references.pmid: 11343927
references.citation: Nat Rev Mol Cell Biol. 2008 Oct;9(10):815-20
references.pmid: 18784729
references.citation: Nat Cell Biol. 2008 May;10(5):538-46
references.pmid: 18408734
references.citation: J Biol Chem. 2008 Jul 18;283(29):19912-21
references.pmid: 18499666
references.citation: Electrophoresis. 2000 Apr;21(6):1037-53
references.pmid: 10786879
references.citation: Yeast. 1998 Jul;14(10):943-51
references.pmid: 9717240
references.citation: Cell. 2002 Jan 11;108(1):109-20
references.pmid: 11792325
references.citation: Curr Opin Chem Biol. 2004 Dec;8(6):610-6
references.pmid: 15556404
references.citation: Cell. 2006 Nov 3;127(3):455-7
references.pmid: 17081966
references.citation: Biochem J. 2003 May 15;372(Pt 1):97-104
references.pmid: 12597774
references.citation: J Mol Biol. 1987 Apr 5;194(3):531-44
references.pmid: 3041007
references.citation: Mol Cell Biol. 1999 Dec;19(12):8660-72
references.pmid: 10567589
references.citation: Nature. 2002 Sep 12;419(6903):135-41
references.pmid: 12226657
references.citation: J Proteome Res. 2008 Sep;7(9):4050-7
references.pmid: 18707152
references.citation: EMBO J. 2004 Oct 1;23(19):3844-53
references.pmid: 15359282
references.citation: Mol Cell. 2005 Apr 1;18(1):1-12
references.pmid: 15808504
references.citation: Mol Cell Biol. 2000 Feb;20(4):1254-62
references.pmid: 10648611
references.citation: J Mol Biol. 1998 Jul 10;280(2):275-86
references.pmid: 9654451
references.citation: Mol Cell Biol. 2005 Jan;25(1):185-96
references.pmid: 15601841
references.citation: Chromosoma. 2007 Feb;116(1):15-20
references.pmid: 17031663
references.citation: J Biol Chem. 2003 Nov 7;278(45):44113-20
references.pmid: 12941945
references.citation: J Cell Sci. 1995 Feb;108 ( Pt 2):475-86
references.pmid: 7768995
references.citation: J Biol Chem. 2001 Sep 21;276(38):35368-74
references.pmid: 11451954
references.citation: Mol Cell. 2004 May 21;14(4):491-500
references.pmid: 15149598
references.citation: Nucleic Acids Res. 1997 Mar 15;25(6):1162-9
references.pmid: 9092625
references.citation: Nat Cell Biol. 2008 May;10(5):547-55
references.pmid: 18408733
references.citation: J Biol Chem. 2002 Dec 6;277(49):47938-45
references.pmid: 12354763
references.citation: Annu Rev Cell Biol. 1987;3:1-30
references.pmid: 2825735