Fig. 7.
Tdp1 activity assays on DNA oligonucleotides with 3′ tetrahydrofuran or 3′ biotin moieties. A. Structure of the abasic site mimic tetrahydrofuran on the DNA 3′ end. B. A 20-mer oligonucleotide containing the tetrahydrofuran moiety on the 3′ end (lane 3, 20-AB) was incubated with wt Tdp1 (lane 4) or the H263A mutant enzyme (lane 5). A corresponding 20-mer with a 3′ hydroxyl group (lane 1, 20-OH) was used as a size marker. The 20-mer with a 3′ phosphate (20-P) was generated by incubating a 20-mer with a 3′ tyrosyl moiety with wt Tdp1 (lane 2). C. Structure of biotin connected to the 3′ end of the DNA by a 15-atom linker. D. Wt Tdp1 (lane 4) or H263A (lane 5) were incubated with a 3′ biotinylated 20-mer DNA (lane 1, 20-biotin) and analyzed on a sequencing gel. To generate size markers (20-OH and 20-P),` a corresponding 3′ phosphorylated 20-mer was labeled with either wt PNK (lane 2) or a 3′ phosphatase minus mutant PNK (PNK−) (lane 3).