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FACT subunit Spt16 controls UVSSA recruitment to lesion-stalled RNA Pol II and stimulates TC-NER

Franziska Wienholz1, Di Zhou1, Yasemin Turkyilmaz1

  • 1Department of Molecular Genetics, Oncode Institute, Erasmus MC, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.

Nucleic Acids Research
|February 5, 2019
PubMed

Insights

UV-Stimulated Scaffold Protein A (UVSSA) is recruited to transcription-blocking DNA lesions independently of Cockayne Syndrome protein A (CSA) and Cockayne Syndrome protein B (CSB). The histone chaperone FACT

Area of Science:

  • Molecular Biology
  • DNA Repair Mechanisms
  • Cellular Biology

Background:

  • Transcription-coupled nucleotide excision repair (TC-NER) removes DNA lesions that stall RNA Polymerase II.
  • Key factors like Cockayne Syndrome protein A (CSA), Cockayne Syndrome protein B (CSB), and UV-Stimulated Scaffold Protein A (UVSSA) initiate TC-NER.
  • The precise recruitment mechanism of these factors to DNA damage sites remains unclear.

Purpose of the Study:

  • To elucidate the recruitment mechanism of UVSSA in transcription-coupled nucleotide excision repair.
  • To investigate the roles of UVSSA domains and interacting proteins in TC-NER.
  • To understand how chromatin modifiers regulate TC-NER pathway steps.

Main Methods:

  • Live-cell imaging to observe UVSSA localization at transcription-blocking DNA lesions (TBLs).
  • Analysis of UVSSA deletion mutants to map functional domains.
  • Quantitative interaction proteomics to identify UVSSA-interacting partners.
  • Depletion studies to assess the impact of Spt16 on TC-NER factor recruitment.

Main Results:

  • UVSSA accumulates at TBLs independently of CSA and CSB.
  • UVSSA's DNA damage recruitment activity is mediated by its VHS and DUF2043 domains.
  • The histone chaperone FACT subunit Spt16 interacts with UVSSA via the DUF2043 domain.
  • Spt16 is recruited to TBLs independently and enhances UVSSA recruitment and TC-NER.
  • Spt16 depletion specifically impacts UVSSA recruitment, not CSB recruitment.

Conclusions:

  • UVSSA recruitment to TBLs is an early, CSA/CSB-independent step in TC-NER.
  • Specific domains of UVSSA mediate DNA damage binding and CSA interaction.
  • Spt16 acts as a chromatin-modulating factor that promotes UVSSA recruitment, highlighting distinct regulatory steps within the TC-NER pathway.

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