Figure 6. Detection of interactions between free and DNA bound hOGG1 and APE1 via gluteraldehide crosslinking.

(A) hOGG1 and APE1 do not strongly interact in the free form. Physiologically relevant concentration of hOGG1 (50 nM) and APE1 (50 – 200 nM) were pre-incubated for 5 min followed by addition of the gluteraldehyde crosslinking agent (50 mM) and a further 20 min incubation. The samples then were resolved using SDS-PAGE with detection by silver staining. No bands corresponding to an APE1-hOGG1 complex were detected. (B) Presence of AP-DNA does not facilitate APE1-hOGG1 interaction. AP-DNA (100 nM) was pre-incubated with hOGG1 (100 nM) for 5 min to form a AP-DNA/hOGG1 complex, followed by addition of APE1 (100 nM). After a 1 min incubation, crosslinking with 50 mM glutaraldehyde was performed for 20 min. The samples were resolved using SDS PAGE silver staining detection. No bands corresponding to APE1-hOGG1 or APE1-hOGG1-APDNA complexes were detected. However, presence of AP-DNA significantly facilitated self-crosslinking of APE1 in the absence of hOGG1. (C) APE1 does not form a stable complex with a reduced hOGG1-APDNA complex. In this experiment designed to test whether APE1 can interact with a highly stable hOGG1-APDNA complex, 100 nM hOGG1 was incubated for 5 min with 100 nM of AP-DNA which was labeled with ³²P on the 5’ end to allow formation of the Schiff base intermediate. The intermediate was reduced by the addition of 25 mM NaBH₄. The reduced complex was then incubated for 1 min with 100 nM APE1 followed by 20 min crosslinking in the presence of 50 mM gluteraldehyde. The samples were resolved on a 10% native polyacrylamide gel with detection by phosphorimaging the ³²P radiolabel. Although a stable hOGG1-APDNA complex was readily detected, no unique bands corresponding to APE1-hOGG1-APDNA complex were observed.