Global Conformational Dynamics of a Y-Family DNA Polymerase during Catalysis
Figure 4
Stopped-flow kinetics of dTTP incorporation into S-2 DNA at 20°C.
Dpo4 mutant•S-2 DNA complexes were reacted with dTTP and the fluorescence was monitored using a stopped-flow apparatus. Donor (green) and acceptor (red) traces are shown for the (A) finger (N70C), (B) palm (S112C), (C) thumb (S207C), and (D) LF (K329C) domains. Data for finger (E49C), palm (S96C and N130C), thumb (K172C), and LF (R267C) residues are shown in Figure S4. Each Dpo4 mutant (Table S1) and S-2 were labeled with Alexa594 and Alexa488, respectively. Notably, some changes in fluorescence upon dTTP binding occurred during the instrument's dead time and the donor and acceptor fluorescence signals at time zero or close to time zero were not recorded.