The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair

J Biol Chem. 2016 Jan 15;291(3):1387-97. doi: 10.1074/jbc.M115.683235. Epub 2015 Nov 30.

Abstract

Cockayne syndrome (CS) is a recessive disorder that results in deficiencies in transcription-coupled nucleotide excision repair (TC-NER), a subpathway of nucleotide excision repair, and cells from CS patients exhibit hypersensitivity to UV light. CS group B protein (CSB), which is the gene product of one of the genes responsible for CS, belongs to the SWI2/SNF2 DNA-dependent ATPase family and has an ATPase domain and an ubiquitin-binding domain (UBD) in the central region and the C-terminal region, respectively. The C-terminal region containing the UBD is essential for the functions of CSB. In this study, we generated several CSB deletion mutants and analyzed the functions of the C-terminal region of CSB in TC-NER. Not only the UBD but also the C-terminal 30-amino acid residues were required for UV light resistance and TC-NER. This region was needed for the interaction of CSB with RNA polymerase II, the translocation of CS group A protein to the nuclear matrix, and the association of CSB with chromatin after UV irradiation. CSB was modified by small ubiquitin-like modifier 2/3 in a UV light-dependent manner. This modification was abolished in a CSB mutant lacking the C-terminal 30 amino acid residues. However, the substitution of lysine residues in this region with arginine did not affect SUMOylation or TC-NER. By contrast, substitution of a lysine residue in the N-terminal region with arginine decreased SUMOylation and resulted in cells with defects in TC-NER. These results indicate that both the most C-terminal region and SUMOylation are important for the functions of CSB in TC-NER.

Keywords: Cockayne syndrome; DNA damage; chromatin; nucleotide excision repair; protein domain; small ubiquitin-like modifier (SUMO); transcription-coupled repair.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Blotting, Western
  • Cell Line
  • DNA Breaks / radiation effects
  • DNA Helicases / chemistry
  • DNA Helicases / genetics
  • DNA Helicases / metabolism*
  • DNA Repair Enzymes / chemistry
  • DNA Repair Enzymes / genetics
  • DNA Repair Enzymes / metabolism*
  • DNA Repair* / radiation effects
  • Fibroblasts / enzymology
  • Fibroblasts / metabolism
  • Fibroblasts / radiation effects
  • Gene Deletion
  • Humans
  • Immunoprecipitation
  • Lysine
  • Mutation
  • Poly-ADP-Ribose Binding Proteins
  • Protein Interaction Domains and Motifs
  • Radiation Tolerance
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Small Ubiquitin-Related Modifier Proteins / metabolism
  • Sumoylation* / radiation effects
  • Transcription, Genetic*
  • Ubiquitins / metabolism
  • Ultraviolet Rays / adverse effects

Substances

  • Poly-ADP-Ribose Binding Proteins
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • SUMO2 protein, human
  • SUMO3 protein, human
  • Small Ubiquitin-Related Modifier Proteins
  • Ubiquitins
  • DNA Helicases
  • ERCC6 protein, human
  • DNA Repair Enzymes
  • Lysine