Nb bsrdi endonuclease

Reporter constructs containing the specified modifications in the 5′-untranslated region of the enhanced green fluorescent protein (EGFP) gene were generated as described previously (40 (link)). Expression vector pZAJ-5C was nicked at tandem sites with the Nb.BsrDI endonuclease (NEB GmbH, Frankfurt am Main, Germany) to generate a 18-nt gap in the transcribed strand followed by annealing and ligation of the specified synthetic oligonucleotides. All oligonucleotides were HPLC-purified and validated by mass spectrometry. Synthetic 18-mer oligonucleotides 5′-CATTGCTTC[THF/S-THF]CTAGCACG containing a tetrahydrofuran AP lesion with either phosphodiester (THF) or phosphorothioate (S-THF) 5′-linkage were from BioSpring GmbH (Frankfurt am Main, Germany). Unmodified reference deoxyribo-oligonucleotide 5′-CATTGCTTCGCTAGCACG was from Eurofins Genomics (Ebersberg, Germany). The oligonucleotide 5′-CATTGC[dT<>dT]CGCTAGCACG containing TT dimer was from TriLink BioTechnologies (San Diego, CA). The oligonucleotide 5′-CATTGCTTC[dG(N²)-AAF]CTAGCACG containing the 3-(deoxyguanosin- N²-yl)-2-acetylaminofluorene adduct was produced as described previously (40 (link)). The oligonucleotide 5′-CATTGCTTCGC[Fluorescein-dT]AGCACG containing C5-fluorescein-dT adduct was from BioSpring GmbH.

Mammalian expression vectors pZAJ-5w-AGC and pZAJ-5c-AGC, containing the 5′-CATTGCTTCGCTAGCACGCATTGC sequence in opposite orientations within the the 5′ untranslated region (5′-UTR) of a gene coding for the enhanced green fluorescent protein (EGFP), were described previously [23] (link). A pair of vectors, containing the same sequences in an arbitrarily chosen non-genic region upstream of the immediate early CMV promoter, was constructed by analogous procedures from the same maternal pZAJ vector.
Site-specific double nicks were produced in either the transcribed or coding DNA strand with the Nb.BsrDI endonuclease (NEB GmbH, Frankfurt am Main, Germany). After melting the excised 18-mers away, the synthetic oligonucleotides (unmodified or containing the specified adducts) were inserted and the nicks sealed by the protocol described previously [24] (link). The presence of dG(C8)-AAF and dG(N²)-AAF in vector DNA was verified by the inhibition of generation of a double-stranded break by the NheI restriction endonuclease; the presence of TT dimer was verified by incision with T4 endonuclease V (Figure S1).