In con trast, the G,U activities and enzymatic www.selleckchem.com/products/z-vad-fmk.html turnovers were very sensitive to sumoylation or SUMO 1 addition in a dose dependent manner. We have measured a G,U turnover rate increased by a factor of 3. 9 for the sumoylated TDG as compared to the non modified TDG, while a 2. 4 and 5. 4 fold increase was observed upon addition of 5 and 10 molar equivalents of SUMO 1, respectively. We have shown in control experiments that the non covalent SUMO 1 effect is highly specific as same amounts of BSA did not induce such a stimulation of TDG and sumoylated TDG glycosylase activities. Furthermore, indeed, free SUMO 1 can also further increase G,T and G,U processivity of sumoy lated TDG unlike BSA.

Finally, the increase in activity of TDG that we postulated based on NMR experiments can be shown to take place under the same experimental conditions as the protein protein and protein DNA interactions, that is in NMR buffer at pH 6. 6. Note that while TDGs processiv ity drops by almost an order of magnitude when using acidic buffers, however, the specific stimulation by sumoylation and free SUMO 1 is clearly detectable and comparable to the one detected under standard experimental conditions. Hence SUMO 1, similarly to the sumoyla tion of TDG, positively acts on the G,U glycosylase activity and also improves albeit weakly the G,T activ ity. Hence, despite a disruption of SBM2 SUMO 1 interactions in presence of DNA or upon SBM2 mutation, SUMO 1 was still able to activate TDG glycosylase activities on both G,T and G,U sub strates in a dose dependent manner suggesting an indirect mechanism where the TDG SUMO 1 interac tion is not directly responsible for the up regulation of glycosylase activity.

SUMO 1 competes with TDG RD for DNA binding Since SUMO 1 does not interact with the TDG C term inal SBM upon SBM mutation or DNA addition, it rather seems that SUMO 1 acts indirectly on TDG activity by an unknown mechanism. We have thus investigated the ability of SUMO 1 to directly interact with DNA and shown a non specific but detectable interaction using NMR spectroscopy and gel shift assays. In this study, we have also demonstrated competi tion between SUMO 1 and TDG RD for DNA binding with EMSA. Here, we demonstrate the ability of SUMO 1 to dis place RD from DNA in a direct competition experiment using NMR methodology.

In presence of an equimolar amount of a double stranded 25 mer DNA substrate containing a G,T mismatch, some weak chemical shift Carfilzomib perturbations of TDG RD were observed and are more pronounced with a 4 fold molar excess of the same sub strate. Adding a 4 fold molar excess of SUMO 1 to the equimolar TDG N, DNA mixture induces a shift of RD resonances towards those for the free RD. This effect concerns resonances for residues comprised in the region from position 75 to 91, indicat ing a partial competition of SUMO 1 with the RD for DNA binding. For the N and C terminal parts of TDG RD, no competition was observed.