Fig. 2.

Gap-filling DNA synthesis and removal of the dRP group by polymerase β in the presence of a DNA trap. Schematic representations of ³²P-labeled DNA substrate (S) and a DNA trap (T) are illustrated above the phosphorimage of the gels. E denotes polymerase β. (a) Gap-filling DNA synthesis by polymerase β in the presence of a DNA trap was examined. The reaction mixture was assembled on ice, either with 60 nM polymerase β and 20 nM 5′-end ³²P-labeled UDG/APE1-treated DNA (lanes 1–3), or with polymerase β and DNA trap (lanes 4, 5). Reactions were initiated by temperature jump and the addition of a mixture of dCTP, DNA trap, and MgCl₂ (lanes 1–3), or dCTP, ³²P-labeled UDG/APE1-treated DNA, and MgCl₂ (lanes 4, 5), respectively. Samples were withdrawn at 10 and 20 s and analyzed. The positions of the ³²P-labeled primer and 1-nt gap-filling product are indicated. (b) For analyzing the dRP lyase activity of polymerase β in the presence of a DNA trap, the reaction mixture was assembled on ice, either with 60 nM polymerase β and 20 nM 3′-end ³²P-labeled UDG/APE1-treated DNA (lanes 1–3), or polymerase β and the trap (lanes 4, 5). Reactions were initiated by temperature jump and addition of a DNA trap (lanes 1–3) or ³²P-labeled substrate (lanes 4, 5), respectively. Samples were withdrawn at 10 and 20 s. The reaction products were stabilized by the addition of NaBH₄, and the reaction products were analyzed. The positions of the ³²P-labeled dRP substrate and the product are indicated.