Figure 4.

Effect of HAP1 upon the AP lyase activity of human OGG1. A limiting amount (5 fmol) of hOGG1 was incubated under standard assay conditions with 50 fmol of the [8-oxoG:C] duplex ³²P-labelled at the 3′-end of the lesion-containing strand and increasing amounts of wild-type HAP1 (lanes 1–6) or HAP1-D210N (lane 7). A 35mer-[U:C] duplex (50 fmol) ³²P-labelled at the 3′-end of the lesion-containing strand was incubated under the same conditions with excess amounts of UDG and endo IV, where indicated (lanes 8–10). Reaction products were separated by denaturing 20% PAGE. Arrows indicate the positions of the 3′-radiolabelled substrate and reaction products as follows: 35 nt, non-cleaved 35mer oligonucleotide; 19 nt, 19mer produced by OGG1 strand nicking activity; *19 nt, 19mer with a 5′-deoxyribose-5-phosphate produced by UDG and endo IV treatment of the 35mer [U:C] duplex. Sequences of the radiolabelled DNA species are shown below the figure.