FIG. 2.

Immunodepletion of the uracil-releasing activity present in A. fulgidus cell extract. Protein extract (240 μg) (in panel A also purified enzyme [0.88 μg]) was treated with different volumes of rabbit antiserum plus reaction buffer in a 500-μl (total volume) mixture, which was followed by centrifugation as described in Materials and Methods. (A) Supernatant following the immunodepletion procedure (25 μl) was incubated for 10 min with [³H]uracil-containing DNA (2,000 dpm; 2 pmol DNA uracil residues) in reaction buffer at 80°C. Symbols: •, cell extract treated with anti-rAfung^H; ○, cell extract treated with control serum; □, rAfung^H′ (rAfung^H following enzymatic removal of the His tag) treated with anti-rAfung^H. Each value is the mean of three independent measurements. The lower graph is an expansion of the 0-to-1 μl part of the upper graph. (B) Supernatant following immunodepletion (5 μl) was incubated with substrate 1 (³²P-labeled 5′-TAGACATTGCCCTCGAGGTAUCATGGATCCGATTTCGACCTCAAACCTAGACGAATTCCG-3′ plus complementary strand; 4 fmol) at 60°C for 10 min in reaction buffer (20 μl). As a result of uracil excision and base-catalyzed phosphodiester bond cleavage (generating a mixture of β- and δ-elimination products; see Fig. 7A), each ³²P-end-labeled oligonucleotide (60 nt) was converted into one ³²P-labeled 20-nt product and one 40-nt unlabeled product. The strand opposite the damage-containing strand is indicated by a dashed line; the sizes of the ³²P-labeled repair intermediates and products are indicated by solid lines. CS, control serum.