Movie S5.

Reinitiation of DNA unwinding by RecQ. A two-color image of a λ DNA molecule containing two unwound regions is shown. The DNA molecule was only partially unwound in a previous experiment and unwinding had apparently halted after several kilobase pairs of unwinding. The dsDNA is stained with Sytox Orange and the two unwound regions are marked with AF488SSB. At ∼15 s into the movie, a solution containing 70 nM RecQ, 60 nM AF488SSB, 1 mM ATP, 1 mM Mg(OAc)2, and 75 nM Sytox Orange is introduced into the flow cell, using the microfluidic injection system shown in Fig. S7. The flowcell is finished filling at ∼47 s, at which point the Sytox Orange in the flow cell starts photobleaching, resulting in decreased background fluorescence. Subsequent continued unwinding (as class I/III events) from the preexisting unwound regions is now evident, until the DNA molecule breaks. The time is indicated in minutes:seconds.