Requirement for human AP endonuclease 1 for repair of 3'-blocking damage at DNA single-strand breaks induced by reactive oxygen species

Tadahide Izumi, Tapas Hazra, Istvan Boldogh, Alan E. Tomkinson, Min S. Park, Shogo Ikeda, Sankar Mitra

Research output: Contribution to journalArticle

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Abstract

The major mammalian apurinic/apyrimidinic (AP) endonuclease (APE1) plays a central role in the DNA base excision repair pathway (BER) in two distinct ways. As an AP endonuclease, it initiates repair of AP sites in DNA produced either spontaneously or after removal of uracil and alkylated bases in DNA by monofunctional DNA glycosylases. Alternatively, by acting as a 3'-phosphoesterase, it initiates repair of DNA strand breaks with 3'-blocking damage, which are produced either directly by reactive oxygen species (ROS) or indirectly through the AP lyase reaction of damage-specific DNA glycosylases. The endonuclease activity of APE1, however, is much more efficient than its DNA 3'-phosphoesterase activity. Using whole extracts from human HeLa and lymphoblastoid TK6 cells, we have investigated whether these two activities differentially affect BER efficiency. The repair of ROS-induced DNA strand breaks was significantly stimulated by supplementing the reaction with purified APE1. This enhancement was linearly dependent on the amount of APE1 added, while addition of other BER enzymes, such as DNA ligase I and FEN1, had no effect. Moreover, depletion of endogenous APE1 from the extract significantly reduced the repair activity, suggesting that APE1 is essential for repairing such DNA damage and is limiting in extracts of human cells. In contrast, when uracil-containing DNA was used as the substrate, the efficiency of repair was not affected by exogenous APE1, presumably because the AP endonuclease activity was not limiting. These results indicate that the cellular level of APE1 may differentially affect repair efficiency for DNA strand breaks but not for uracil and AP sites in DNA.

Original languageEnglish (US)
Pages (from-to)1329-1334
Number of pages6
JournalCarcinogenesis
Volume21
Issue number7
StatePublished - 2000

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Single-Stranded DNA Breaks
Endonucleases
Reactive Oxygen Species
DNA Breaks
Uracil
DNA
DNA Repair
DNA Glycosylases
DNA-(Apurinic or Apyrimidinic Site) Lyase
Lyases
Cell Extracts
DNA Damage
Enzymes

ASJC Scopus subject areas

  • Cancer Research

Cite this

Requirement for human AP endonuclease 1 for repair of 3'-blocking damage at DNA single-strand breaks induced by reactive oxygen species. / Izumi, Tadahide; Hazra, Tapas; Boldogh, Istvan; Tomkinson, Alan E.; Park, Min S.; Ikeda, Shogo; Mitra, Sankar.

In: Carcinogenesis, Vol. 21, No. 7, 2000, p. 1329-1334.

Research output: Contribution to journalArticle

Izumi, Tadahide ; Hazra, Tapas ; Boldogh, Istvan ; Tomkinson, Alan E. ; Park, Min S. ; Ikeda, Shogo ; Mitra, Sankar. / Requirement for human AP endonuclease 1 for repair of 3'-blocking damage at DNA single-strand breaks induced by reactive oxygen species. In: Carcinogenesis. 2000 ; Vol. 21, No. 7. pp. 1329-1334.
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