Figure 5.

Analysis of the products of the dOgg1 repair reaction. (A) Mechanism of strand cleavage. The 34mer carrying a single 8-OH-Gua residue was ³²P-labeled at its 5′ end and hybridized with a complementary sequence carrying a cytosine opposite the lesion. Incubation were performed with 10 ng of dOgg1, yOgg1 or Fpg protein for 15 min at 37°C. When indicated, reaction mixtures were then incubated with 10 ng of either endonuclease III (Nth) or endonuclease IV (Nfo). The products were separated by denaturing 20% PAGE. P1, P2 and P3 are the products described in the Results. (B) NaBH₄-mediated trapping assay. The 34mer carrying a single 8-OH-Gua residue was labeled at its 5′-end and hybridized with a complementary sequence carrying a cytosine opposite the lesion. Fifty nanograms of either Fpg, yOgg1 or dOgg1 proteins were allowed to react for 20 min at 37°C with the ³²P-labeled 34mer duplex DNA, containing a single 8-OH-Gua.C, in the presence of 50 mM of either NaCl or NaBH₄. The products of the reactions were separated using 15% SDS–PAGE.