The non covalent SUMO binding capa city of TDG is also negatively affected by DNA binding through the TDG N terminal region. It is this non covalent SUMO 1 binding which stimulates CBP dependent transcriptional activation and is involved in TDG translocation to PML oncogenic domains, implicating its ability to bind sumoylated PML or other sumoylated selleck kinase inhibitor proteins found within this nuclear compart ment. For both SUMO 1 conjugation and intermolecular SUMO 1 binding, the N terminal domain of TDG was found to be targeted in the modification of TDG func tion in BER. We have previously reported that the regu latory domain, located in the N terminus of TDG, provides an additional non sequence or mis match specific DNA binding activity and furthermore established dynamic intramolecular interactions with the core catalytic domain.

This interface is altered in the presence of a DNA substrate. Moreover, the conformation of the regulatory domain modulates the TDG glycosylase activity and enzymatic turnover in a mismatch dependent manner. Here we describe the effects on the conformational dynamics of TDG, and in particular on the regulatory domain, of SUMO 1 conju gation on the one hand and non covalent SUMO 1 bind ing on the other. The mechanism of stimulation of TDG glycosylase activity by SUMO 1 is described. Results SUMO 1 conjugation to TDG affects the C terminal domain conformation but not the N terminal region of TDG The uniformly 15N labeled TDG protein conjugated on lysine 330 to SUMO 1 was produced in E. coli as described.

The conjugation site was verified using as a negative control the TDG K330A mutant under the same conditions for protein production. In this latter control case only the non modified TDG K330A protein was isolated after purification as checked by MALDI TOF MS and denaturing gel electrophoresis. Thus sumoylation of TDG under these condi tions indeed only occurs on lysine 330. In our previous NMR study, we have shown that the TDG protein exhibits broad lines on the 15N 1H HSQC spectrum concerning the large majority of its residues and that only the N and C terminus resonances are detectable due to their high degree of flexibility in solu tion. We have also shown critical conformational dynamics for the regulatory domain of the N terminus.

This region, coinciding with a functional domain implicated in speci fic G,T excision, adopts a residual structure in the context of the isolated N terminus and undergoes a dra matic conformational and dynamic change in the con text of the entire protein leading to the disappearance broadening of corresponding resonances. Anacetrapib The disap pearance of resonances was shown to be due to intra molecular RD CAT interactions. As for the unconjugated TDG protein, the acquisition of a 15N 1 H HSQC spectrum on SUMO modified TDG leads to the detection of random coil regions.