Characterization of O4-alkyldT Lesions

All chemicals, if not specifically described, were from Sigma-Aldrich (St. Louis, MO, USA) or EMD Millipore, and all enzymes, unless otherwise noted, were obtained from New England Biolabs (Ipswich, MA, USA). 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was obtained from TCI America (Portland, OR, USA) and [γ-³²P]ATP was purchased from Perkin Elmer (Piscataway, NJ, USA). All unmodified oligodeoxyribonucleotides (ODNs) were from Integrated DNA Technologies (IDT, Coralville, IA, USA). The 12-mer ODNs harboring a site-specifically incorporated O⁴-alkyldT were synthesized using conventional phosphoramidite chemistry, as described previously (14 (link)). The identified and purities of all the lesion-harboring ODNs were confirmed by liquid chromatography-mass spectrometry (LC-MS) and tandem MS (MS/MS) analyses prior to their insertion into double-stranded plasmids.