Escherichia coli uracil dna glycosylase

DNA oligonucleotides were synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). Deoxynucleotide 5′-triphosphates (dNTPs) were obtained from Sigma-Aldrich (St. Louis, MO, USA). T4 polynucleotide kinase and terminal deoxynucleotidyl transferase were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Radionucleotides [γ-³²P] ATP (3000 Ci/mmol) and Cordycepin 5′-triphosphate 3′-[α-³²P] (5000 Ci/mmol) were purchased from Perkin Elmer Inc. (Boston, MA, USA). Micro Bio-Spin 6 chromatography columns were acquired from Bio-Rad Laboratories (Hercules, CA, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), l(+)-glutamine, and 0.25% trypsin-EDTA were purchased from Thermo Fisher Scientific (Waltham, MA, USA). All standard chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Thermo Fisher Scientific (Waltham, MA, USA). Escherichia coli uracil-DNA glycosylase (UDG) was purchased from New England BioLabs Inc. (Ipswich, MA, USA). Purified recombinant pol β dRP lyase mutant (K72A and H34G) and pol β 8 kD domain proteins were generous gifts from Dr Samuel H. Wilson at the National Institute of Environmental Health Sciences/National Institutes of Health. Purified recombinant human APE1, wild-type (WT) pol β, FEN1 and DNA ligase I (LIG I) were expressed and purified as described previously (14 (link),18 (link),32 (link)).