title: UV-induced RPA phosphorylation is increased in the absence of DNA polymerase eta and requires DNA-PK.
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 eta (poleta). Poleta is required for error-free bypass of UV-induced cyclobutane pyrimidine dimers; in the absence of poleta 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 poleta 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 poleta can be induced by addition of tetracycline, expression of poleta 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 poleta compared to when poleta 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 poleta.
authors.affiliation: DNA Damage Response Laboratory, Department of Biochemistry and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Galway City, Ireland.
authors.forename: Séverine
authors.initials: S
authors.lastname: Cruet-Hennequart
authors.forename: Seamus
authors.initials: S
authors.lastname: Coyne
authors.forename: Macdara T
authors.initials: MT
authors.lastname: Glynn
authors.forename: Gregory G
authors.initials: GG
authors.lastname: Oakley
authors.forename: Michael P
authors.initials: MP
authors.lastname: Carty
mesh_terms: D002453:Cell Cycle / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000096:biosynthesis / Q000528:radiation effects; D004249:DNA Damage / Q000235:genetics; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000172:deficiency* / Q000378:metabolism; D004307:Dose-Response Relationship, Radiation; D005347:Fibroblasts / Q000166:cytology; D015971:Gene Expression Regulation, Enzymologic; D006657:Histones / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation / Q000528:radiation effects; D011494:Protein Kinases / Q000378:metabolism; D051716:Replication Protein A / Q000378:metabolism*; D013752:Tetracycline / Q000378:metabolism; D014466:Ultraviolet Rays; D000099270:Y-Family DNA Polymerases
chemical_list: D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D004247:DNA; D051747:DNA-Activated Protein Kinase; D004259:DNA-Directed DNA Polymerase; D006657:Histones; D009687:Nuclear Proteins; D011494:Protein Kinases; D051716:Replication Protein A; D013752:Tetracycline; C468783:H2AX protein, human; C112156:NBN protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases