Loading of Polβ onto AP sites by Ape protein. (A) Binding to AP sites in different sequence contexts. Purified human Ape protein (0.55 pmol) or Polβ (0.55 pmol) were incubated with the indicated 5′-labeled AP substrates in reactions containing either 4 mM EDTA or 10 mM MgCl₂ (Mg²⁺), and the uncomplexed DNA (lowest bands) and protein DNA complexes (upper bands) resolved by electrophoresis in a nondenaturing gel (22). Binding substrates were 23-F, a 23-bp duplex DNA containing a tetrahydrofuran residue (10); 18-AP, an 18-bp oligonucleotide containing an AP site generated by uracil excision (10); and 51-AP, a 51-bp duplex oligonucleotide containing an AP site generated by uracil excision (13). (B) Presence of Ape and Polβ in complexes. Binding reactions with EDTA or Mg²⁺ were carried out with Ape or Polβ and the 51-AP substrate as described above, then Ape-specific (αApe) or Polβ-specific (αβ-pol) antisera were added. The complexes containing Ape or Polβ (middle bands) were resolved from the antibody-supershifted complexes (top bands) by electrophoresis in nondenaturing gels. In no case was significant material retained in the wells of the gels.

Interaction of Ape and Polβ in the yeast two-hybrid system. (Left) Schematic of plasmids present in indicator strain Y190 (20). Plasmid pAS1–Ape encodes the Gal4_DB–Ape fusion; pACT–Polβ encodes the Gal4_AD–Polβ fusion; pSE1111 encodes a Gal4_AD-Snf4 fusion; pSE1112 encodes a Gal4_DB-Snf1 fusion (19). (Center) Indicator plate for β-galactosidase expression from a GAL promoter-lacZ fusion and His⁺ selection from a GAL promoter-HIS3 fusion. (Right) Quantitation of β-galactosidase expression by a fluorescent assay. Ape-DBD is the Gal4_DB–Ape fusion, Polβ-AD the Gal4_AD–Polβ fusion. Six independent cotransformants of pAS1-Ape and pACT-Polβ were assayed for β-galactosidase activity; single isolates of the other transformants were assayed. An S. cerevisiae lacZ⁺ strain (obtained from R. Brennan and R. H. Schiestl, Harvard School of Public Health, Boston) was assayed as a positive control.

Activation of Polβ dRp excision activity by Ape. (A) A duplex oligonucleotide containing an AP site at position 22 was cleaved with a catalytic amount of E. coli endonuclease IV, then incubated for the indicated times with purified human Polβ (9 fmol) and varying amounts of purified human Ape protein (0, 8, 80, or 800 fmol). After the incubation, the substrate bearing 5′-dRp (upper band) and the product after dRp excision (lower band) were resolved in a denaturing gel. OH⁻, substrate hydrolyzed with NaOH; X, the 5′-dRp substrate before incubation; 15, 5′-dRp substrate incubated 15 min with 800 fmol of Ape alone. (B) Quantitation of dRp excision. The gel in A was subjected to scanning densitometry and the ratio of the substrate (upper band in A) to product (lower band in A) used to calculate the excision of dRp in 25-min reactions.