TI - Reversal of 3-meT measured by HPLC analysis . AB - We considered it highly likely that the observed AlkB -mediated release of a 3-meT -induced replication block was caused by an actual oxidative demethylation of 3-meT to thymine . However , it could not be formally excluded that the result of the oxidative action of the human and bacterial AlkB proteins on 3-meT is not thymine , but rather another product which allows bypass by T7 DNA polymerase . To study this , the 3-meT containing oligonucleotide was digested to nucleosides by enzymatic hydrolysis with nuclease P1 and alkaline phosphatase , and the resulting nucleosides analysed by reverse-phase HPLC . Five peaks were detected in the chromatogram ; four major peaks corresponding to the unmodified nucleosides adenosine , cytidine , guanosine and thymidine , and a minor peak corresponding to the single 3-mdT residue (Figure 3A) . However , when the oligonucleotide was treated with hABH2 prior to HPLC analysis of its constituent nucleosides , the peak corresponding to 3-mdT was barely visible , while the thymidine peak increased slightly , indicating reversal of the 3-meT lesion to the normal base thymine (Figure 3B) . In accordance with the results of the primer extension experiments , the highest degree of repair was observed in the case of hABH2 , and we were only able to see partial reversal of the 3-meT lesion with AlkB or hABH3 ( data not shown ) . Our HPLC analysis confirms the major finding from the primer extension experiments , i.e.that 3-meT is a SUBstrate for AlkB , and also demonstrates that the lesion is repaired by demethylation .