Stimulation of TDG by UV-DDB.

Abstract

DNA methylation plays a key role in epigenetics, with 60-80% of CpG sites containing 5-methylcytosine. Base excision repair (BER) is suggested to be the main pathway involved in DNA demethylation. 5-formylctyosine (5fC), an oxidized moiety of methylated cytosine, is recognized and removed by thymine DNA glycosylase (TDG) to generate an abasic site. TDG binds avidly to abasic sites and is product inhibited. We have previously shown that UV-DDB plays a role in BER and stimulates OGG1, APE1, and MUTYH. In this study, we investigate the mechanism by which UV-DDB stimulates the activity of TDG. Kinetic assays showed that UV-DDB was able to increase the turnover number of TDG excising 5fC from DNA by 14-fold. Electrophoretic mobility shift assays revealed the dissociation of TDG from abasic sites with increasing concentrations of UV-DDB. An additional band on the gel indicates the dissociation of TDG is caused by the formation of a transient TDG-UV-DDB co-complex. Atomic force microscopy will be utilized to verify the formation of this co-complex. Using single molecule fluorescent experiments, we saw TDG interact with DNA containing 5fC with lifetimes of 93.4 and 23.4 seconds respectively. We also saw TDG move on undamaged DNA with a lifetime of 29.2 seconds. These results indicate that TDG cleaves the 5fC and stays bound for an extended time at the generated abasic site. TDG moves for a shorter amount of time on non-specific DNA. The catalytically crippled mutant, N140A, binds to DNA containing 5fC with a lifetime of 1.9 seconds and undamaged DNA with a lifetime of 2.1 seconds. These results indicate that mutating N140 interferes with damage recognition by TDG. Further studies will be done to study the behavior of wild type and mutant TDG in the presence of UV-DDB at the single molecule level.