UV-induced RPA phosphorylation is increased in the absence of DNA polymerase η and requires DNA-PK

Séverine Cruet-Hennequart, Seamus Coyne, Macdara T. Glynn, Gregory G. Oakley, Michael P. Carty

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

Signaling from arrested replication forks plays a role in maintaining genome stability. We have investigated this process in xeroderma pigmentosum variant cells that carry a mutation in the POLH gene and lack functional DNA polymerase η (polη). Polη is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of polη in XPV cells, DNA replication is arrested at sites of UV-induced DNA damage, and mutagenic bypass of lesions is ultimately carried out by other, error-prone, DNA polymerases. The present study investigates whether polη expression influences the activation of a number of UV-induced DNA damage responses. In a stably transfected XPV cell line (TR30-9) in which active polη can be induced by addition of tetracycline, expression of polη determines the extent of DNA double-strand break formation following UV-irradiation. UV-induced phosphorylation of replication protein A (RPA), a key DNA-binding protein involved in DNA replication, repair and recombination, is increased in cells lacking polη compared to when polη is expressed in the same cell line. To identify the protein kinase responsible for increased UV-induced hyperphosphorylation of the p34 subunit of RPA, we have used NU7441, a specific small molecule inhibitor of DNA-PK. DNA-PK is necessary for RPA p34 hyperphosphorylation, but DNA-PK-mediated phosphorylation is not required for recruitment of RPA p34 into nuclear foci in response to UV-irradiation. The results demonstrate that activation of a UV-induced DNA damage response pathway, involving phosphorylation of RPA p34 by DNA-PK, is enhanced in cells lacking polη.

Original languageEnglish (US)
Pages (from-to)491-504
Number of pages14
JournalDNA Repair
Volume5
Issue number4
DOIs
StatePublished - Apr 8 2006

Keywords

  • DNA-PK
  • Polη
  • RPA
  • UV damage
  • XPV

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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