Figure 4.

NMR studies also indicate that the excised base is released from enzyme–product complexes. All four panels shown an identical ¹⁵N-TROSY spectrum for TDG^cat bound to AP-DNA (black peaks); the sample was prepared by adding TDG^cat to purified abasic DNA (AP-DNA). The red peaks in panels A–C are ¹⁵N-TROSY data for enzyme–product complexes resulting from TDG^cat action on various substrates, including G·hmU (A), G·T (B) and G·U (C). The absence of substantial chemical shift changes indicates that the excised base is released from the enzyme–product complex. (D) The red peaks are ¹⁵N-TROSY data for TDG_cat bound to DNA containing a G·U^F base pair; U^F is a dU analog that flips into the active site but cannot be cleaved by TDG. Substantial chemical shift changes are observed for most backbone ¹⁵N-¹H resonances; the DNA differs at only one nucleotide (AP site versus U^F, see inset). Samples contained 0.25 mM ¹⁵N-labeled TDG^cat, 0.30 mM DNA, 5 mM Tris–HCl pH 7.5, 0.2 M NaCl, 0.2 mM DTT, 0.2 mM EDTA, 10% D₂O.