Fig. 3.

Gap-filling DNA synthesis and removal of the dRP group steps by polymerase β are concurrent. Schematic representations of a ³²P-DNA substrate labeled at both ends (S) and the DNA trap (T) are illustrated above the phosphorimage of the gels. E denotes polymerase β. Double-labeled 34-bp DNA was prepared by annealing a 5′-end labeled 15-mer oligonucleotide and a 3′-end labeled 19-mer oligonucleotide to their complementary 34-mer DNA strand. The 19-mer oligonucleotide also contained a 5′-end phosphate and uracil. The duplex DNA was pretreated with UDG, resulting in a single-nucleotide gapped DNA with 3′-OH and 5′-dRP groups at the margins and radiolabels on both ends. Gap-filling DNA synthesis and dRP lyase reactions were performed by polymerase β in the presence of excess DNA trap. The repair reaction mixture was assembled on ice, either with polymerase β and ³²P-labeled substrate (a), or polymerase βand DNA trap (b). Reactions were initiated by temperature jump and the addition of a mixture of dCTP, DNA trap, and MgCl₂ (a), or dCTP, ³²P-labeled substrate DNA, and MgCl₂ (b), respectively. Samples were withdrawn at 10 and 20 s and analyzed. The positions of the ³²P-Iabeled primer, 1-nt gap-filling DNA synthesis product, ³²P-labeled dRP substrate and the dRP lyase product are indicated.