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

Mol Cell Proteomics. 2015 May;14(5):1419-34. doi: 10.1074/mcp.O114.044792. Epub 2015 Mar 9.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Amino Acid Sequence
  • BRCA1 Protein / chemistry
  • BRCA1 Protein / genetics
  • BRCA1 Protein / metabolism
  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Cell Line, Tumor
  • Chromatin Assembly Factor-1 / chemistry
  • Chromatin Assembly Factor-1 / genetics
  • Chromatin Assembly Factor-1 / metabolism
  • DNA Damage
  • DNA Replication
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Endodeoxyribonucleases / chemistry
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism
  • Gene Expression
  • Genomic Instability
  • Humans
  • Hydroxyurea / pharmacology*
  • Lysine / chemistry
  • Lysine / metabolism*
  • Molecular Sequence Data
  • Nuclear Proteins / chemistry
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Nucleic Acid Synthesis Inhibitors / pharmacology*
  • Osteoblasts / cytology
  • Osteoblasts / drug effects*
  • Osteoblasts / metabolism
  • Protein Interaction Mapping
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism*
  • Signal Transduction
  • Small Ubiquitin-Related Modifier Proteins / chemistry
  • Small Ubiquitin-Related Modifier Proteins / genetics
  • Small Ubiquitin-Related Modifier Proteins / metabolism*
  • Sumoylation
  • Trans-Activators / chemistry
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Transcription Factors

Substances

  • ATRIP protein, human
  • Adaptor Proteins, Signal Transducing
  • BRCA1 Protein
  • BRCA1 protein, human
  • CNOT8 protein, human
  • Carrier Proteins
  • Chromatin Assembly Factor-1
  • DNA-Binding Proteins
  • MDC1 protein, human
  • Nuclear Proteins
  • Nucleic Acid Synthesis Inhibitors
  • RMI1 protein, human
  • Recombinant Fusion Proteins
  • SUMO2 protein, human
  • Small Ubiquitin-Related Modifier Proteins
  • Trans-Activators
  • Transcription Factors
  • Eme1 protein, human
  • Endodeoxyribonucleases
  • Lysine
  • Hydroxyurea