Nucleobases obtained by N1-methylation of purines and N3-methylation of pyrimidines.

Repair of 3-meT lesions by hABH2 catalysed oxidative demethylation. The 3-meT containing oligonucleotide was incubated in the absence (solid line) or presence (dashed line) of hABH2, then degraded enzymatically to its constituent nucleosides by treatment with nuclease P1 and calf intestine alkaline phosphatase, and, finally, subjected to reverse-phase HPLC. The identity of the peaks corresponding to 2′-deoxycytidine (dC), 2′-deoxyguanosine (dG), 2′-deoxythymidine (dT), 2′-deoxyadenosine (dA), and 3-methyl-2′-deoxythymidine (3mdT) was inferred from comparison of their elution times with those of the corresponding reference standards.

Inability of 1-meG and 3-meT, and corresponding nucleosides, to stimulate AlkB-mediated uncoupled decarboxylation of 2-oxoglutarate. Reaction mixtures containing AlkB and [5-¹⁴C]2-oxoglutarate, and 400 μM of the indicated compounds were incubated for 10 min at 37°C. Remaining [5-¹⁴C]2-oxoglutarate was precipitated with 2,4,dinitrophenylhydrazine, and the [1-¹⁴C]succinate in the supernatant was measured by scintillation counting. Error bars represent the range between duplicate measurements. ‘Vehicle’ indicates the addition of an amount of DMSO equivalent to that added with the bases and nucleosides.

Repair of replication blocking lesions by AlkB proteins. (A) Schematic outline of the experiments. ³²P-end-labelled (indicated by asterisk) 15mer oligonucleotide was extended by T7 DNA polymerase on a 24mer oligonucleotide template. (B) Release of 3-meT-induced extension block by AlkB proteins. The 3-meT containing template was subjected to repair by the indicated amounts of human or bacterial AlkB proteins, and subsequently used as template in a primer extension experiment. The primer extension products were analysed by denaturing polyacrylamide analysis and phophorimaging. (C) Quantification of the data shown in (B). The degree of bypass was calculated as the intensity of the 25 nt band, relative to the sum of the 18 and 25 nt bands. (D) Induction of extension block by DMS treatment of template. The 24mer oligonucleotide (without 3-meT) was incubated with the indicated concentrations of DMS, and, subsequently, used as template in a primer extension experiment. (E) Release of DMS-induced extension block by AlkB proteins. The 24mer oligonucleotide (without 3-meT) was treated with 100 mM DMS, then incubated with the indicated amounts of AlkB proteins, and, finally, used as template for primer extension. (F) Quantification of the data shown in (E). The amount of radioactivity present in the 25 nt band has been expressed relative to the sum of the radioactivity contained in all the bands in each lane.

AlkB-mediated repair of 1-meG in chemically methylated tRNA. [³H]MNU-treated 24mer RNA oligonucleotide (A) or [¹⁴C]MeI-treated E.coli tRNA from (B) was incubated in the absence (filled circles) or presence (open circles) of AlkB, then enzymatically degraded to nucleosides, which were subjected by reverse-phase HPLC. Fractions were collected, and the radioactivity present in the fractions determined by scintillation counting. Arrows indicate the elution times of internal nucleoside reference standards corresponding to the modified bases.