Fig. 4. RecT binding promotes unwinding of dsDNA. (A) Supercoiled pHV2900OB DNA (30 µM) was relaxed by incubation with 4 U of wheat germ topoisomerase I for 20 min at 37°C. Then, RecT was added to the reaction mixtures (30 µl) at the concentrations indicated and incubated for a further 45 min at 37°C in buffer T. The reaction products were deproteinized, separated by electrophoresis in a 1% agarose gel and revealed by SYBR green staining. Unreacted supercoiled pHV2900OB DNA is in lane a. The positions of supercoiled and relaxed DNA molecules are indicated. (B) pHV2900OB DNA treated exactly as described above, with 1.67 µM RecT (lane a) or no RecT (lane b). The deproteinized reaction products were subjected to two- dimensional chloroquine gel electrophoresis. Chloroquine concentrations used were 0.66 µM and 2 µM in the first and second dimension, respectively. Position of the nicked circle is indicated by an arrow-head.