Figure 4.

The 9-1-1 complex specifically stimulates the endonuclease activity of APE 1 in vitro. The APE 1 incision assay was performed as described in ‘Materials and Methods’. Reactions were stopped by adding an equal volume of formamide-dye solution and products were analyzed on a 10% denaturing polyacrylamide gel. (A) An APE 1 reaction mixture (10 μl) contained (besides all components described in ‘Materials and Methods’) ³²P-5′-labeled 100 bp duplex oligonucleotide (50 fmol see Figure 1B), APE 1 (2 fmol). Reactions were incubated for 20 min at 37°C with the indicated amounts of the 9-1-1 complex. (B) As A but with the blocked substrate (50 fmol). (C) Quantification of the stimulation of APE 1 endonuclease cleavage by the 9-1-1 complex on the substrate with free ends (closed circles) and with the ends blocked with biotin (open circles). The values represent the mean of three independent experiments. The error bars correspond to the standard error of the mean.