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. 2011 Nov 14;40(5):2000–2009. doi: 10.1093/nar/gkr933

Figure 2.

Figure 2.

Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. (A) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1− (RHP357) and tdp1−nth1− (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. (B) Udg activity in the nth1− and tdp1−nth1− extracts. The nth1− and tdp1−nth1− extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.