Supplementary Figure 2: OGG1 glycosylase/lyase, MUTYH glycosylase, and APE1 endonuclease activities in the absence or presence of UV-DDB, Related to Methods and Fig. 1.

(a) Comparison of APE1 incision activity on 37 bp DNA with a THF moiety (left) and an authentic abasic site (right). AP37 substrate is the product of a reaction between dU37 and Uracil DNA glycosylase. (b) AP lyase activity of OGG1 on 8-oxoG37(G:C) with increasing amount of OGG1, as indicated. DNA products were separated by denaturing polyacrylamide electrophoresis. (c) Chemical structure of 8-oxoG:C. (d) Effect of UV-DDB concentration on stimulation of OGG1 incision. 8-oxoG37(G:C) was incubated with OGG1 and/or increasing amounts of UV-DDB at 37°C for 1.5hrs and separated by denaturing polyacrylamide electrophoresis. (e) Quantification of (d). Percent of total DNA that was incised by OGG1 plotted as a function of UV-DDB concentration. Data shown as the mean of three experiments ± s.d. (f) Effect of UV-DDB on stimulation of OGG1 incision. 8-oxoG37(G:C) was incubated with OGG1 at 37°C for 2hrs and UV-DDB (16nM) was added. Incised DNA was separated by denaturing polyacrylamide electrophoresis. (g) Quantification of (f). Percent of total DNA that was incised by OGG1 plotted as a function of time. Data shown as the mean of three experiments ± s.d. (h) Effect of APE1 and UV-DDB on stimulation of OGG1 incision. 8-oxoG37(G:C) was incubated with OGG1 only, OGG1+APE1, or OGG1+APE1+UV-DDB for 2hrs at 37°C and separated by denaturing polyacrylamide electrophoresis. (i) Quantification of (h). Percent of total DNA that was incised by OGG1 plotted as a function of time. Data shown as the mean of three experiments ± s.d. (** p< 0.01). (j) Effect of UV-DDB concentration on stimulation of MUTYH glycosylase activity. 8-oxoG37(G:A) was incubated with MUTYH and/or increasing amount of UV-DDB for 80mins at 37°C. The reaction was immediately stopped by adding 2X loading dye with 0.1M NaOH followed by heating 95°C for 5mins then quickly chilling on ice for 5mins. (k) Quantification of (j). Percent of total DNA that was incised by MUTYH plotted as a function of UV-DDB concentration. Data shown as the mean of three experiments ± s.d. (l) Effect of UV-DDB concentration on stimulation of APE1 incision. THF37 was incubated with OGG1 and/or increasing amounts of UV-DDB at RT for 1hr and separated by denaturing polyacrylamide electrophoresis. (m) Quantification of (l). Percent of total DNA that was incised by APE1 plotted as a function of UV-DDB concentration. Data shown as the mean of three experiments ± s.d. (n) Stimulation of APE1 activity by UV-DDB. THF-containing DNA (200 nM) was incubated with APE1 (1 nM) in the absence (-) (Lanes 1-3) or presence (+) (Lanes 4-6) of UV-DDB (50 nM) for the periods indicated (i.e., 2, 5 and 10 min). Aliquots (4.5 μl) were removed for analysis at the indicated times. The reaction was terminated by addition of an equal volume of DNA gel loading buffer. The reaction products were analyzed as described in methods. The migration positions of the substrate and product are indicated. The results shown are representative of three experiments. (o) Quantification of (n). Solid line represents mean value of three experiments at 10 mins, normalized to APE1 alone. Black circles represent APE1 activity in the presence of UV-DDB, normalized to reference APE1 activity (right, set to 1.0).