DNA polymerase ε associates with the elongating form of RNA polymerase II and nascent transcripts

Anna K. Rytkönen, Tomi Hillukkala, Markku Vaara, Miiko Sokka, Maarit Jokela, Raija Sormunen, Heinz Peter Nasheuer, Tamar Nethanel, Gabriel Kaufmann, Helmut Pospiech, Juhani E. Syväoja

Research output: Contribution to journalArticlepeer-review

Abstract

DNA polymerase ε co-operates with polymerases α and δ in the replicative DNA synthesis of eukaryotic cells. We describe here a specific physical interaction between DNA polymerase ε and RNA polymerase II, evidenced by reciprocal immunoprecipitation experiments. The interacting RNA polymerase II was the hyperphosphorylated IIO form implicated in transcriptional elongation, as inferred from (a) its reduced electrophoretic mobility that was lost upon phosphatase treatment, (b) correlation of the interaction with phosphorylation of Ser5 of the C-terminal domain heptapeptide repeat, and (c) the ability of C-terminal domain kinase inhibitors to abolish it. Polymerase ε was also shown to UV crosslink specifically α-amanitin-sensitive transcripts, unlike DNA polymerase α that crosslinked only to RNA-primed nascent DNA. Immunofluorescence microscopy revealed partial colocalization of RNA polymerase IIO and DNA polymerase ε, and immunoelectron microscopy revealed RNA polymerase IIO and DNA polymerase ε in defined nuclear clusters at various cell cycle stages. The RNA polymerase IIO-DNA polymerase ε complex did not relocalize to specific sites of DNA damage after focal UV damage. Their interaction was also independent of active DNA synthesis or defined cell cycle stage.

Original languageEnglish
Pages (from-to)5535-5549
Number of pages15
JournalFEBS Journal
Volume273
Issue number24
DOIs
StatePublished - Dec 2006

Keywords

  • DNA polymerase ε
  • DNA replication
  • Immunoelectron microscopy
  • Nucleotide excision repair
  • RNA polymerase II

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