Активность ДНК-гликозилаз суперсемейства FPG/NEI в интермедиатах транскрипции

Abstract

Oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. Repair of oxidative base lesions is initiated by DNA glycosylases. For example, bacterial Fpg and Nei DNA glycosylases excise oxidized purines and pyrimidines, respectively, from DNA. Their human homologs, NEIL1 and NEIL2, have been reported to show preference towards oxidized lesions in DNA bubbles. From these observations, it had been hypothesized that NEIL proteins may be involved in the repair of lesions in DNA bubbles generated during transcription. However, it is not presently clear how NEILs would behave on bubbles more closely resembling transcription intermediates (e. g., containing the RNA strand), and bacterial homologs Fpg and Nei had never been investigated with bubble substrates. We have studied excision of either 8-oxoguanine (8-oxoG) or 5,6-dihydrouracil (DHU) by E. coli Fpg and Nei and human NEIL1 and NEIL2 from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases (6 to 30), Dloops with DNA or RNA and from complexes with RNA polymerase. Fpg, NEIL1 and NEIL2 efficiently excised DHU located inside a bubble. Fpg and NEIL1 was generally more active than NEIL2 in excision of 8-oxoG from ssDNA and bubbles. Nei, on the other hand, was active only on DHU located in dsDNA (either perfect duplex or DNA/DNA D-loop). Fpg and NEIL1 also have shown activity in D-loops with RNA. The activity of Fpg was observed in pre-assembled transcriptional complexes with E. coli RNA polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex.