Modulation of DNA-dependent protein kinase activity in chlorambucil-treated cells

Attila Bacsi, Subbaraj Kannan, Myung Soog Lee, Tapas K. Hazra, Istvan Boldogh

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

DNA-dependent protein kinase (DNA-PK) is activated in a two-step process whereby the Ku heterodimer first binds to the DNA double-strand breaks (dsbs) and then the DNA-PK catalytic subunit (cs) is recruited to form a repair complex. Oxidative stress is simultaneously generated along with DNA damage by ionizing radiation or chemotherapeutic agents whose impact on the DNA-PK activity has not previously been investigated. Here we show that the DNA damage-induced kinase activity of DNA-PK was modulated by oxidative stress, which was induced along with DNA dsbs in chlorambucil (Cbl)-exposed cells. Pretreatment with the antioxidants, 2(3)-t-butyl-4-hydroxyanisole or N-acetyl-l-cysteine enhanced the amount of DNA-PKcs phosphorylated at threonine 2609 (DNA-PKpThr2609) at the DNA dsbs and DNA-PK activity. Conversely, oxidative stress induced by l-buthionine (SR)-sulfoximine or glucose oxidase decreased the DNA-PK activity in Cbl-exposed cells. In addition, DNA-PKpThr2609 was poorly detectable at the site of DNA dsbs, as shown by colocalization to DNA-end-binding pH2AX or p53BP1. There was no change in the protein levels of DNA-PKcs, Ku70, or Ku86. Data from these studies provide the first evidence that oxidative stress effects posttranslational modification and assembly of DNA-PK complex at DNA dsbs, and thereby repair of DNA dsbs.

Original languageEnglish (US)
Pages (from-to)1650-1659
Number of pages10
JournalFree Radical Biology and Medicine
Volume39
Issue number12
DOIs
StatePublished - Dec 15 2005

Keywords

  • DNA dsbs repair
  • DNA-PK
  • Free radicals
  • Oxidative stress

ASJC Scopus subject areas

  • Medicine(all)
  • Toxicology
  • Clinical Biochemistry

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