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Clustered DNA damage, where two or more lesions are located proximally to each other, is frequently induced by ionizing radiation. Individual base lesions within a cluster are repaired by base excision repair. In this study we addressed the question of how thymine glycol (Tg) within a cluster would affect the repair of opposing lesions by human cell extracts. We have found that Tg located opposite to an abasic site does not affect cleavage of this site by apurinic/apyrimidinic (AP) endonuclease. However, Tg significantly compromised the next step of the repair. Although purified DNA polymerase beta was able to incorporate the correct nucleotide (dAMP) opposite to Tg, the rate of incorporation was reduced by 3-fold. Tg does not affect 5'-sugar phosphate removal by the 2-deoxyribose-5-phosphate (dRP) lyase activity of DNA polymerase beta, but further processing of the strand break by purified DNA ligase III was slightly diminished. In agreement with these findings, although an AP site located opposite to Tg was efficiently incised in human cell extract, only a limited amount of fully repaired product was observed, suggesting that such clustered DNA lesions may have a significantly increased lifetime in human cells compared with similar single-standing lesions.

Original publication

DOI

10.1074/jbc.M212068200

Type

Journal article

Journal

J Biol Chem

Publication Date

14/03/2003

Volume

278

Pages

9378 - 9381

Keywords

Carbon-Oxygen Lyases, Chromatography, High Pressure Liquid, DNA, DNA Damage, DNA Polymerase beta, DNA Repair, DNA-(Apurinic or Apyrimidinic Site) Lyase, Dose-Response Relationship, Drug, HeLa Cells, Humans, Oligonucleotides, Osmium Tetroxide, Ribosemonophosphates, Thymine, Time Factors