Transcription coupled DNA repair protein UVSSA binds to DNA and RNA: Mapping of nucleic acid interaction sites on human UVSSA

Arch Biochem Biophys. 2023 Feb:735:109515. doi: 10.1016/j.abb.2023.109515. Epub 2023 Jan 6.

Abstract

Transcription-coupled repair (TCR) is a dedicated pathway for the preferential repair of bulky transcription-blocking DNA lesions. These lesions stall the elongating RNA-polymerase II (RNAPII) triggering the recruitment of TCR proteins at the damaged site. UV-stimulated scaffold protein A (UVSSA) is a recently identified cofactor which is involved in stabilization of the TCR complex, recruitment of DNA-repair machinery and removal/restoration of RNAPII from the lesion site. Mutations in UVSSA render the cells TCR-deficient and have been linked to UV-sensitive syndrome. Human UVSSA is a 709-residue long protein with two short conserved domains; an N-terminal (residues 1-150) and a C-terminal (residues 495-605) domain, while the rest of the protein is predicted to be intrinsically disordered. The protein is well conserved in eukaryotes, however; none of its homologs have been characterized yet. Here, we have purified the recombinant human UVSSA and have characterized it using bioinformatics, biophysical and biochemical techniques. Using EMSA, SPR and fluorescence-based methods, we have shown that human UVSSA interacts with DNA and RNA. Furthermore, we have mapped the nucleic acid binding regions using several recombinant protein fragments containing either the N-terminal or the C-terminal domains. Our data indicate that UVSSA possesses at least two nucleic acid binding regions; the N-terminal domain and a C-terminal tail region (residues 606-662). These regions, far apart in sequence space, are predicted to be in close proximity in structure-space suggesting a coherent interaction with target DNA/RNA. The study may provide functional clues about the novel family of UVSSA proteins.

Keywords: Nucleotide excision repair; Protein RNA interactions; Transcription-coupled repair; UV-Sensitive syndrome; UVSSA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carrier Proteins / metabolism
  • DNA / metabolism
  • DNA Damage
  • DNA Repair
  • Humans
  • Nucleic Acids*
  • RNA Polymerase II / genetics
  • RNA Polymerase II / metabolism
  • RNA*
  • Receptors, Antigen, T-Cell / genetics
  • Receptors, Antigen, T-Cell / metabolism
  • Transcription, Genetic

Substances

  • RNA
  • Carrier Proteins
  • Nucleic Acids
  • DNA
  • RNA Polymerase II
  • Receptors, Antigen, T-Cell
  • UVSSA protein, human