Mutsumi Kobayashi

Enzymatic Repair of 5-Formyluracil I. EXCISION OF 5-FORMYLURACIL SITE-SPECIFICALLY INCORPORATED INTO OLIGONUCLEOTIDE SUBSTRATES BY AlkA PROTEIN (Escherichia coli 3-METHYLADENINE DNA GLYCOSYLASE II)

5-Formyluracil (fU) is a major thymine lesion produced by reactive oxygen radicals and photosensitized oxidation. We have previously shown that fU is a potentially mutagenic lesion due to its elevated frequency to mispair with guanine. Therefore, fU can exist in DNA as a correctly paired fU:A form or an incorrectly paired fU:G form. In this work, fU was site-specifically incorporated opposite A in oligonucleotide substrates to delineate the cellular repair mechanism of fU paired with A. The repair activity for fU was induced inEscherichia coli upon exposure toN-methyl-N′-nitro-N-nitrosoguanidine, and the induction was dependent on the alkA gene, suggesting that AlkA (3-methyladenine DNA glycosylase II) was responsible for the observed activity. Activity assay and determination of kinetic parameters using purified AlkA and defined oligonucleotide substrates containing fU, 5-hydroxymethyluracil (hU), or 7-methylguanine (7mG) revealed that fU was recognized by AlkA with an efficiency comparable to that of 7mG, a good substrate for AlkA, whereas hU, another major thymine methyl oxidation products, was not a substrate. 1H and 13C NMR chemical shifts of 5-formyl-2′-deoxyuridine indicated that the 5-formyl group caused base C-6 and sugar C-1′ to be electron deficient, which was shown to result in destabilization of the N-glycosidic bond. These features are common in other good substrates for AlkA and are suggested to play key roles in the differential recognition of fU, hU, and intact thymine. Three mammalian repair enzymes for alkylated and oxidized bases cloned so far (MPG, Nth1, and OGG1) did not recognize fU, implying that the mammalian repair activity for fU resided on a yet unidentified protein. In the accompanying paper (Terato, H., Masaoka, A., Kobayashi, M., Fukushima, S., Ohyama, Y., Yoshida, M., and Ide, H.,J. Biol. Chem. 274, 25144–25150), possible repair mechanisms for fU mispaired with G are reported.

DNA strand breaks induced by ionizing radiation on Rubrobacter radiotolerans, an extremely radioresistant bacterium

Rubrobacter radiotolerans is the most radioresistant bacterium showing 16kGy as D37 against gamma-rays. However mechanisms of the radioresistance have been still unclear. To clarify the post-irradiating events in the cell, we investigated DNA strand breaks which is the most major lesion induced by ionizing radiation. The neutral sucrose density gradient centrifugation analysis showed that size of the chromosomal DNA gradually decreased with irradiation dose. However, the frequency of DNA strand breaks after ionizing irradiation was significantly lower than those reported for other eubacteria. The reduced DNA sizes were not recovered during the post-irradiating cultivation. These results suggest that in this organism DNA protection from damage is mainly involved in the radioresistance, but not DNA repair activities.