Pre-steady-state fluorescence analysis of damaged DNA transfer from human DNA glycosylases to AP endonuclease APE1

• Kinetic analysis of DNA transfer from DNA glycosylases to AP endonuclease was performed.
• The direct protein–protein interactions in the transient ternary complex were found.
• The “flip-flop” exchange of DNA strands in the ternary complex DNA/DNA glycosylase/AP endonuclease was hypothesized.
• Mechanism of coordination of the initial stages of BER was elucidated.

BackgroundDNA glycosylases remove the modified, damaged or mismatched bases from the DNA by hydrolyzing the N-glycosidic bonds. Some enzymes can further catalyze the incision of a resulting abasic (apurinic/apyrimidinic, AP) site through β- or β,δ-elimination mechanisms. In most cases, the incision reaction of the AP-site is catalyzed by special enzymes called AP-endonucleases.MethodsHere, we report the kinetic analysis of the mechanisms of modified DNA transfer from some DNA glycosylases to the AP endonuclease, APE1. The modified DNA contained the tetrahydrofurane residue (F), the analogue of the AP-site. DNA glycosylases AAG, OGG1, NEIL1, MBD4cat and UNG from different structural superfamilies were used.ResultsWe found that all DNA glycosylases may utilise direct protein–protein interactions in the transient ternary complex for the transfer of the AP-containing DNA strand to APE1.ConclusionsWe hypothesize a fast “flip-flop” exchange mechanism of damaged and undamaged DNA strands within this complex for monofunctional DNA glycosylases like MBD4cat, AAG and UNG. Bifunctional DNA glycosylase NEIL1 creates tightly specific complex with DNA containing F-site thereby efficiently competing with APE1. Whereas APE1 fast displaces other bifunctional DNA glycosylase OGG1 on F-site thereby induces its shifts to undamaged DNA regions.General significanceKinetic analysis of the transfer of DNA between human DNA glycosylases and APE1 allows us to elucidate the critical step in the base excision repair pathway.