FIGURE 5.

TDG^ΔSUMO is regulated similarly to TDG^WT. A, TDG is destroyed at a slow rate compared with Cdt1, and SUMOylation does not affect the total TDG destruction rate. Destruction of TDG^WT or TDG^ΔSUMO was triggered with 5 ng/μl MMS-damaged plasmid and measured as in Fig. 4B. B, quantification of the rate of destruction of Cdt1 and different forms of TDG in A. C, mutation of the SUMOylation site (K364A) eliminates modification of TDG to a higher molecular weight and increases the affinity of TDG for DNA. TDG^WT or TDG^ΔSUMO was added to extract that was subsequently added to linear, MMS-damaged DNA immobilized on magnetic beads. Methyl ubiquitin was included to allow monoubiquitylation on multiple sites but to prevent polyubiquitylation and destruction. Loading control in total extract was as in Fig. 4B; loading control for DNA-bound fraction was Orc2. D, TDG^ΔSUMO is destroyed during DNA repair. Buffer, MMS-damaged plasmid, or undamaged plasmid was added to egg extract, and TDG levels were measured as in Fig. 4B. E, TDG^ΔSUMO destruction depends on the proteasome. TDG^ΔSUMO destruction was measured as in Fig. 4B in the presence of DMSO (vehicle) or 1 mm MG132. F, depletion of Cdt2 stabilizes TDG^ΔSUMO. Egg extract was mock-depleted with preimmune serum or immunodepleted with anti-Cdt2 antiserum, and TDG^ΔSUMO destruction was measured as in Fig. 4B. Loading controls for A and E were as in Fig. 4C. Loading controls for D and F were as in Fig. 4B. Error bars, S.D.