The ring fragmentation product of thymidine C5-hydrate when present in DNA is repaired by the Escherichia coli Fpg and Nth proteins.

Various forms of oxidative stress, including gamma-radiolysis and UV irradiation, result in the formation of damaged bases. (5R)-Thymidine C5-hydrate is one of several modified nucleosides produced from thymidine under these conditions. N-(2-Deoxy-beta-D-erythro-pentofuranosyl)-N-3-[(2R)-hydroxyisobutyric acid]urea or alphaRT is the respective fragmentation product formed from (5R)-thymidine C5-hydrate upon hydrolysis. This modified nucleoside has potential mutagenic or lethal properties. No enzymatic activity responsible for the removal of alphaRT has been identified. We report here that when present in DNA, alphaRT is a substrate for two purified enzymes from Escherichia coli involved in the repair of oxidized bases: the Nth and the Fpg proteins. The Fpg protein removes the alphaRT lesion more efficiently than the Nth protein. This is the first example of efficient excision of a ring-opened form of a pyrimidine by the Fpg protein. The high efficacy of the Fpg protein suggests that it is likely to be involved in vivo in the excision of alphaRT. The kinetics of the reaction of the Fpg protein with DNA containing alphaRT suggest substrate inhibition. Duplex oligodeoxynucleotides containing alphaRT positioned opposite T, dG, dC, and dA were cleaved efficiently by both enzymes, although the profiles of activity of the two enzymes were different. The Nth enzyme preferentially excises alphaRT when opposite a dG, followed by alphaRT.dA, alphaRT. T, and alphaRT.dC. For the Fpg protein, the order is alphaRT.dC >/= alphaRT.dG approximately alphaRT.T > alphaRT.dA. Moreover, we show that human cell extract exhibits an activity that excises alphaRT from an oligonucleotide, suggesting that human homologues of the Nth and/or Fpg proteins could be involved in repair of this lesion in human cells.