title: System-wide Analysis of SUMOylation Dynamics in Response to Replication Stress Reveals Novel Small Ubiquitin-like Modified Target Proteins and Acceptor Lysines Relevant for Genome Stability.
abstract: Genotoxic agents can cause replication fork stalling in dividing cells because of DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells. The developed methodology enabled single step purification of His10-SUMO-2 conjugates under denaturing conditions with high yield and high purity. Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. After 2 h of hydroxyurea treatment, 10 proteins were up-regulated for SUMOylation and two proteins were down-regulated for SUMOylation, whereas after 24 h, 35 proteins were up-regulated for SUMOylation, and 13 proteins were down-regulated for SUMOylation. A site-specific approach was used to map over 1000 SUMO-2 acceptor lysines in target proteins. The methodology is generic and is widely applicable in the ubiquitin field. A large subset of these identified proteins function in one network that consists of interacting replication factors, transcriptional regulators, DNA damage response factors including MDC1, ATR-interacting protein ATRIP, the Bloom syndrome protein and the BLM-binding partner RMI1, the crossover junction endonuclease EME1, BRCA1, and CHAF1A. Furthermore, centromeric proteins and signal transducers were dynamically regulated by SUMOylation upon replication stress. Our results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress. Ultimately, our study reveals how a post-translational modification is able to orchestrate a large variety of different proteins to integrate different nuclear processes with the aim of dealing with the induced DNA damage.
authors.affiliation: From the ‡Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands;
authors.forename: Zhenyu
authors.initials: Z
authors.lastname: Xiao
authors.affiliation: From the ‡Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands;
authors.forename: Jer-Gung
authors.initials: JG
authors.lastname: Chang
authors.affiliation: From the ‡Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands;
authors.forename: Ivo A
authors.initials: IA
authors.lastname: Hendriks
authors.affiliation: §Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
authors.forename: Jón Otti
authors.initials: JO
authors.lastname: Sigurðsson
authors.affiliation: §Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3B, 2200 Copenhagen, Denmark.
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
authors.affiliation: From the ‡Department of Molecular Cell Biology, Leiden University Medical Center, 2300 RC Leiden, the Netherlands; vertegaal@lumc.nl.
authors.forename: Alfred C O
authors.initials: AC
authors.lastname: Vertegaal
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019313:BRCA1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D045744:Cell Line, Tumor; D056504:Chromatin Assembly Factor-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D042822:Genomic Instability; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology*; D008239:Lysine / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology*; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D025941:Protein Interaction Mapping; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D058207:Sumoylation; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors
chemical_list: C521194:ATRIP protein, human; D048868:Adaptor Proteins, Signal Transducing; D019313:BRCA1 Protein; C492913:BRCA1 protein, human; C489780:CNOT8 protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056504:Chromatin Assembly Factor-1; D004268:DNA-Binding Proteins; C413163:MDC1 protein, human; D009687:Nuclear Proteins; D019384:Nucleic Acid Synthesis Inhibitors; C501527:RMI1 protein, human; D011993:Recombinant Fusion Proteins; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C507947:Eme1 protein, human; D004706:Endodeoxyribonucleases; D008239:Lysine; D006918:Hydroxyurea