FIG. 3.

Stimulation of the 3′→5′ exonuclease and 3′-phosphodiesterase activities of Apn2 by PCNA. Apn2 (5 nM) was incubated with DNA substrate (20 nM) in a standard reaction mixture containing 0 mM NaCl for 10 min at 30°C. Reactions were carried out in the absence (lanes 2, 5, and 8) or presence (lanes 3, 6, and 9) of PCNA (50 nM). No protein (np) was added to the reactions in lanes 1, 4, and 7. Reaction products were analyzed on an 8 M urea-10% polyacrylamide gel. The 3′→5′ exonuclease activity was assayed on a 44- and 75-nt partial DNA duplex containing a 5′ ³²P-labeled strand with a 3′ recessed terminus (A). Phosphodiesterase activity was tested on a 35- and 70-nt partial DNA duplex containing a 5′ ³²P-labeled strand with a recessed 3′-PG terminus (B). The AP endonuclease assay was examined on a 75-nt DNA duplex containing a single abasic site at position 31 on the 5′ ³²P-labeled strand (C). The asterisks indicate the radioactively labeled termini.