title: Sumoylation of the Rad1 nuclease promotes DNA repair and regulates its DNA association.
abstract: The Saccharomyces cerevisiae Rad1-Rad10 complex is a conserved, structure-specific endonuclease important for repairing multiple types of DNA lesions. Upon recruitment to lesion sites, Rad1-Rad10 removes damaged sequences, enabling subsequent gap filling and ligation. Acting at mid-steps of repair, the association and dissociation of Rad1-Rad10 with DNA can influence repair efficiency. We show that genotoxin-enhanced Rad1 sumoylation occurs after the nuclease is recruited to lesion sites. A single lysine outside Rad1's nuclease and Rad10-binding domains is sumoylated in vivo and in vitro. Mutation of this site to arginine abolishes Rad1 sumoylation and impairs Rad1-mediated repair at high doses of DNA damage, but sustains the repair of a single double-stranded break. The timing of Rad1 sumoylation and the phenotype bias toward high lesion loads point to a post-incision role for sumoylation, possibly affecting Rad1 dissociation from DNA. Indeed, biochemical examination shows that sumoylation of Rad1 decreases the complex's affinity for DNA without affecting other protein properties. These findings suggest a model whereby sumoylation of Rad1 promotes its disengagement from DNA after nuclease cleavage, allowing it to efficiently attend to large numbers of DNA lesions.
authors.affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA Programs in Biochemistry, Cell, and Molecular Biology, Weill Cornell Graduate School of Medical Sciences, New York, NY 10065, USA.
authors.forename: Prabha
authors.initials: P
authors.lastname: Sarangi
authors.affiliation: Department of Biology, Masaryk University, Brno 62500, Czech Republic.
authors.forename: Zdenka
authors.initials: Z
authors.lastname: Bartosova
authors.affiliation: Department of Biology, Masaryk University, Brno 62500, Czech Republic.
authors.forename: Veronika
authors.initials: V
authors.lastname: Altmannova
authors.affiliation: Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
authors.forename: Cory
authors.initials: C
authors.lastname: Holland
authors.affiliation: National Centre for Biomolecular Research, Masaryk University, Brno 62500, Czech Republic.
authors.forename: Melita
authors.initials: M
authors.lastname: Chavdarova
authors.affiliation: Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA Division of Radiation Biology, Department of Radiation Oncology, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
authors.forename: Sang Eun
authors.initials: SE
authors.lastname: Lee
authors.affiliation: Department of Biology, Masaryk University, Brno 62500, Czech Republic National Centre for Biomolecular Research, Masaryk University, Brno 62500, Czech Republic International Clinical Research Center, St. Anne's University Hospital in Brno, Brno 60200, Czech Republic.
authors.forename: Lumir
authors.initials: L
authors.lastname: Krejci
authors.affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA zhaox1@mskcc.org.
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Zhao
mesh_terms: D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology; D008239:Lysine / Q000378:metabolism; D009154:Mutation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D058207:Sumoylation; D044767:Ubiquitin-Protein Ligases / Q000502:physiology
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D029701:Saccharomyces cerevisiae Proteins; C436836:Siz2 protein, S cerevisiae; D004247:DNA; D044767:Ubiquitin-Protein Ligases; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D004720:Endonucleases; C069266:RAD1 protein, S cerevisiae; C436837:Siz1 protein, S cerevisiae; D045643:DNA Repair Enzymes; D008239:Lysine
references.citation: Anticancer Res. 2010 Sep;30(9):3223-32
references.pmid: 20944091
references.citation: J Biochem. 2003 Apr;133(4):415-22
references.pmid: 12761287
references.citation: Proc Natl Acad Sci U S A. 2005 Mar 29;102(13):4777-82
references.pmid: 15738391
references.citation: Curr Biol. 2005 Apr 12;15(7):616-23
references.pmid: 15823533
references.citation: Biochem Soc Trans. 2009 Jun;37(Pt 3):495-510
references.pmid: 19442243
references.citation: Mol Cell Biol. 2014 Mar;34(5):778-93
references.pmid: 24344201
references.citation: Nat Struct Mol Biol. 2011 Jul 10;18(8):920-6
references.pmid: 21743457
references.citation: Genes Dev. 2004 Sep 15;18(18):2283-91
references.pmid: 15371342
references.citation: Nucleic Acids Res. 2012 Sep 1;40(17):8325-35
references.pmid: 22730299
references.citation: DNA Repair (Amst). 2004 Mar 4;3(3):335-41
references.pmid: 15177048
references.citation: DNA Repair (Amst). 2011 Jul 15;10(7):781-91
references.pmid: 21612988
references.citation: Methods Mol Biol. 2009;590:265-77
references.pmid: 19763510
references.citation: Nucleic Acids Res. 2011 Feb;39(3):781-94
references.pmid: 20929870
references.citation: Mol Cell Biol. 2007 Sep;27(17):6153-62
references.pmid: 17591698
references.citation: Sci Signal. 2009 May 26;2(72):ra24
references.pmid: 19471022
references.citation: EMBO J. 2002 Mar 15;21(6):1456-64
references.pmid: 11889051
references.citation: Annu Rev Biochem. 2008;77:229-57
references.pmid: 18275380
references.citation: Nat Cell Biol. 2006 Nov;8(11):1284-90
references.pmid: 17013376
references.citation: Cell. 2006 Nov 3;127(3):509-22
references.pmid: 17081974
references.citation: Annu Rev Biochem. 2004;73:355-82
references.pmid: 15189146
references.citation: Cell. 1996 Sep 6;86(5):811-22
references.pmid: 8797827
references.citation: Mol Cell. 2012 Aug 10;47(3):444-56
references.pmid: 22749529
references.citation: Mol Cell Biol. 2006 Feb;26(3):1135-41
references.pmid: 16428464
references.citation: Genes Dev. 2001 Sep 1;15(17):2238-49
references.pmid: 11544181
references.citation: J Biol Chem. 2001 Apr 20;276(16):12654-9
references.pmid: 11124955
references.citation: Biochem J. 2005 Oct 15;391(Pt 2):325-33
references.pmid: 15975089
references.citation: Nucleic Acids Res. 2012 Jul;40(13):e101
references.pmid: 22457069
references.citation: Mol Cell Proteomics. 2009 Jun;8(6):1382-90
references.pmid: 19240082
references.citation: Proc Natl Acad Sci U S A. 2006 Jun 13;103(24):8953-8
references.pmid: 16751265
references.citation: Mol Cell. 2008 May 9;30(3):325-35
references.pmid: 18471978
references.citation: EMBO J. 1997 Nov 3;16(21):6559-73
references.pmid: 9351836
references.citation: Curr Opin Cell Biol. 2009 Apr;21(2):237-44
references.pmid: 19230642
references.citation: J Biol Chem. 2001 Jun 15;276(24):21664-9
references.pmid: 11259410
references.citation: PLoS One. 2009;4(4):e5267
references.pmid: 19384408
references.citation: J Biol Chem. 2010 Apr 23;285(17):12803-12
references.pmid: 20190278
references.citation: Nat Struct Mol Biol. 2011 Aug 14;18(9):1015-9
references.pmid: 21841787
references.citation: Chromosoma. 2011 Apr;120(2):109-27
references.pmid: 21369956
references.citation: DNA Repair (Amst). 2011 Dec 10;10(12):1243-51
references.pmid: 21968059
references.citation: Mol Cell. 2005 Jul 1;19(1):123-33
references.pmid: 15989970
references.citation: J Mol Biol. 2008 Feb 8;376(1):221-31
references.pmid: 18155241
references.citation: Nucleic Acids Res. 2010 Aug;38(14):4708-21
references.pmid: 20371517
references.citation: Nature. 2002 Sep 12;419(6903):135-41
references.pmid: 12226657
references.citation: J Biol Chem. 1998 Nov 20;273(47):31541-6
references.pmid: 9813069
references.citation: Proc Natl Acad Sci U S A. 2002 Oct 15;99(21):13669-74
references.pmid: 12368472
references.citation: EMBO J. 2013 Feb 6;32(3):461-72
references.pmid: 23299942
references.citation: J Biol Chem. 1998 Dec 11;273(50):33111-4
references.pmid: 9837874
references.citation: J Biol Chem. 1996 May 3;271(18):10821-6
references.pmid: 8631896
references.citation: Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10718-22
references.pmid: 8855246
references.citation: Cell. 2011 Sep 30;147(1):158-72
references.pmid: 21962513
references.citation: Acta Histochem. 2011 Jul;113(4):409-15
references.pmid: 20546858
references.citation: Cold Spring Harb Perspect Biol. 2013 Oct 01;5(10):a012609
references.pmid: 24086042
references.citation: Mol Cell Biol. 2004 May;24(10):4151-65
references.pmid: 15121837
references.citation: EMBO J. 2002 Jun 3;21(11):2833-41
references.pmid: 12032096
references.citation: Nucleic Acids Res. 2013 May 1;41(10):5341-53
references.pmid: 23571759
references.citation: Annu Rev Genet. 2004;38:233-71
references.pmid: 15568977
references.citation: EMBO J. 2002 Apr 15;21(8):2045-53
references.pmid: 11953324
references.citation: J Biol Chem. 1995 Oct 20;270(42):24638-41
references.pmid: 7559571
references.citation: DNA Repair (Amst). 2010 Jun 4;9(6):718-26
references.pmid: 20382573
references.citation: Gene. 2000 Jan 11;241(2):193-204
references.pmid: 10675030
references.citation: J Biol Chem. 1995 Jun 2;270(22):12973-6
references.pmid: 7768886
references.citation: Mol Cell Biol. 2007 Sep;27(18):6433-45
references.pmid: 17636031
references.citation: J Biol Chem. 1993 Dec 15;268(35):26391-9
references.pmid: 8253764
references.citation: EMBO J. 2013 Apr 17;32(8):1155-67
references.pmid: 23531881
references.citation: J Biol Chem. 2009 Mar 20;284(12):7733-45
references.pmid: 19129197
references.citation: Mol Cell. 2012 Feb 10;45(3):422-32
references.pmid: 22285753
references.citation: J Biol Chem. 2014 Apr 11;289(15):10308-10317
references.pmid: 24567323
references.citation: Nature. 2008 Oct 2;455(7213):689-92
references.pmid: 18716619
references.citation: Biomolecules. 2012 Sep 04;2(3):376-88
references.pmid: 24926426
references.citation: Annu Rev Biochem. 2008;77:259-87
references.pmid: 18518821
references.citation: J Biol Chem. 2013 Jul 19;288(29):20918-20926
references.pmid: 23749995
references.citation: Cell. 2012 Nov 9;151(4):807-820
references.pmid: 23122649
references.citation: Nature. 2009 Dec 17;462(7275):886-90
references.pmid: 20016594
references.citation: Bioessays. 2009 Mar;31(3):315-21
references.pmid: 19260026
references.citation: Yeast. 1998 Jun 30;14(9):847-52
references.pmid: 9818722
references.citation: Methods Mol Biol. 2009;497:3-16
references.pmid: 19107407
references.citation: Mol Cell Biol. 2003 May;23(10):3487-96
references.pmid: 12724407
references.citation: Science. 1994 Sep 30;265(5181):2082-5
references.pmid: 8091230
references.citation: Cell. 2011 Oct 14;147(2):459-74
references.pmid: 21963094
references.citation: DNA Repair (Amst). 2004 Jan 5;3(1):1-12
references.pmid: 14697754
references.citation: Mutat Res. 2000 Jun 30;451(1-2):13-24
references.pmid: 10915862
references.citation: Nature. 2009 Dec 17;462(7275):935-9
references.pmid: 20016603