Fig. 3. RecT binding entails unstacking of ssDNA but not dsDNA. Sensitivity of thymine residues to KMnO₄ in ssDNA (lanes c–h and m–o) or dsDNA (lanes p–u) was monitored as a function of RecT (lanes c–h and p–u) or RecA concentrations (lanes m–o). The indicated enzyme concentrations were incubated with 1.7 µM ³²P-labeled ssDNA or 3.4 µM ³²P-labeled dsDNA in 75 µl of reaction mixtures, for 20 min at 37°C and 10 min at 25°C. The protein–DNA complexes were reacted with 0.6 mM KMnO₄ for 1 min at 25 °C, treated with piperidine as described in Materials and methods, and the DNA products were separated in a 20% urea–polyacrylamide gel. KMnO₄-untreated controls: ssDNA (lane O) and ssDNA reacted with piperidine (lane P). Lanes C, Y, G and R are standard Maxam–Gilbert sequencing reactions. Arrows indicate the positions of thymines.