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A more recent version of this article appeared on March 7, 2003
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M212068200v1
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Papers In Press, published online ahead of print January 8, 2003
J. Biol. Chem, 10.1074/jbc.M212068200
Submitted on November 26, 2002
Revised on January 6, 2003
Accepted on January 7, 2003

Mode of inhibition of short-patch base excision repair by thymine glycol within clustered DNA lesions

Helen Budworth and Grigory L. Dianov

Radiation and Genome Stability Unit, Medical Research Council, Didcot, Oxfordshire OX11 0RD

Corresponding Author: g.dianov{at}har.mrc.ac.uk

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 AP endonuclease. However, Tg significantly compromised the next step of the repair. Although purified DNA polymerase b 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 dRP lyase activity of DNA polymerase b, but further processing of the strand break by purified DNA ligase III was slightly diminished. In agreement with these findings, while 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 life-time in human cells compared to similar single-standing lesions.


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