UNG-directed repair of U•G mispairs in circular duplex substrates. (A) Schematic for construction and repair of M13-U•G substrates. A 56-mer complementary oligonucleotide carrying a single U was annealed to ss M13mp18 to create a U•G mismatch within the Hin dIII restriction site (left); extended with Phusion polymerase to produce a nicked-circular duplex M13-U•G substrates (center); and repaired to create a functional Hin dIII site (right). (B) Reactions demonstrating repair of M13-U•G substrates by purified components of the base excision repair pathway, analyzed by agarose gel electrophoresis. Shown are unrepaired, undigested M13-U•G substrates; and substrates treated with UNG, APE1 and pol β and then digested with Hin dIII and Bme 1580 I, as indicated. Arrows at left indicate undigested nicked circles, and unrepaired and repaired products; schematic at right diagrams products of Hin dIII and Bme 1580 I digestion.