Generation of reporter plasmids containing defined base modifications in the DNA strand of choice

Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

“…In support of this hypothesis, it was shown that the magnitude of the inhibition of transcription in genetically manipulated host cells extremely well correlates with the OGG1 protein amounts and the actual 8-oxoG excision efficiencies (30). Moreover, suppression of transcription appeared to be a common feature of several BER substrates besides 8-oxoG; and in all cases investigated so far the respective specific DNA glycosylases (either mono- or bifunctional) have been clearly implicated ((30,31,32) and unpublished results). All these observations strongly indicate that some common post-excision BER intermediate plays a critical role for the inhibition of transcription in cells.…”

“…Double incisions were generated in the non-transcribed DNA strand of the EGFP gene with the Nt.Bpu10I nicking endonuclease (Thermo Fisher Scientific, Bonn, Germany), after which synthetic oligonucleotides with or without modifications were incorporated into the vector DNA by a straightforward strand-exchange procedure (31). Completeness of the strand exchange reactions was monitored by the inhibition of formation of covalently closed DNA in the absence of polynucleotide kinase, as described previously (31).…”

Excision of Uracil from Transcribed DNA Negatively Affects Gene Expression

“…1A) was achieved by nicking one strand of the reporter gene twice with the nicking endonuclease Nt.Bpu10I or Nb.Bpu10I and swapping the excised 18-nt fragment for a synthetic oligonucleotide as described previously (19). Reporter plasmids containing a single thymine mispaired with guanine were obtained by the same methodology that was used for the construction of vectors with a single U:G mismatch.…”

“…After incubation at 37°C for 60 min, the reactions were stopped by adding SDS to 0.1% and heating at 50°C for 3 min, followed by addition of DNA loading dye and electrophoresis in agarose gels containing ethidium bromide (0.5 mg/liter). Control reactions with uracil-DNA glycosylase and endonuclease IV of Escherichia coli were performed as described previously (19), but the incubation time was increased to 1 h. DNA strand cleavage was determined from the relative intensities of DNA bands by the GelDoc TM XRϩ molecular imager and Image Lab TM software (Bio-Rad) and adjusted for the 2.4-fold difference in the fluorescence yield between the covalently closed and nicked circular DNA (21).…”

“…Human Cells-To investigate the consequences of a single uracil located in a transcribed region of a gene on transcriptional output, we incorporated synthetic oligonucleotides containing a single deoxyuracil into the EGFP coding sequence of the pEGFP-mODC-ZA expression vector using a methodology described previously (19). The oligonucleotides were inserted into either the transcribed or the non-transcribed strand of the gene, with single uracils placed opposite an adenine (U:A) or a guanine (U:G) (Fig.…”