In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts (original) (raw)

Abstract

Base excision repair (BER) is a major cellular repair mechanism that corrects a broad range of DNA lesions (for a review, see 1). BER deals with DNA damage generated not only by environmental genotoxins, like ionizing radiation, alkylating agents and oxidative reagents, but also by endogeneously produced oxygen radicals and other reactive species. Therefore, its correct functioning is very important for genome stability and cell viability (2,3). The primary pathway for BER involves the recognition by a DNA glycosylase of the damaged base followed by cleavage of the _N_-glycosyl bond to generate an apurinic/apyrimidinic (AP) site. The AP site is then recognized by an endonuclease. The major AP endonucleases cleave hydrolytically the phosphodiester bond on the 5′-side of the AP site. A phosphodiesterase then excises the generated 5′-deoxyribose phosphate terminus to leave a single nucleotide gap. This gap can then be filled by a DNA polymerase (polymerase β in mammalian cells) and the nick sealed by a DNA ligase. In eukaryotes, an alternative BER pathway that involves the replacement of more than a single residue is also present (4–6). Repair synthesis, which is dependent upon proliferating cell nuclear antigen (PCNA), occurs on the 3′-side of the damaged residue and involves the replacement of two to six nucleotides. It is likely that these two repair mechanisms have evolved to repair structurally distinct lesions.

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Authors and Affiliations

  1. DNA Repair Unit, CSTA Laboratory, Istituto Nazionale Ricerca Cancro, Genova, Italy
    Guido Frosina & Enrico Cappelli
  2. Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanita, Roma, Italy
    Paola Fortini & Eugenia Dogliotti

Authors

  1. Guido Frosina
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  2. Enrico Cappelli
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  3. Paola Fortini
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  4. Eugenia Dogliotti
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Editor information

Editors and Affiliations

  1. University of Dundee, Dundee, UK
    Daryl S. Henderson

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© 1999 Humana Press Inc.

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Frosina, G., Cappelli, E., Fortini, P., Dogliotti, E. (1999). In Vitro Base Excision Repair Assay Using Mammalian Cell Extracts. In: Henderson, D.S. (eds) DNA Repair Protocols. Methods in Molecular Biology™, vol 113. Humana Press. https://doi.org/10.1385/1-59259-675-4:301

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