Figure 3
From: oriD structure controls RepD initiation during rolling-circle replication
DNA nick-religation activity by RepD. (A) Upper trace (solid line) shows rotational position of the magnetic tweezers and the lower trace (filled circles) shows the bead height as a function of time. The 10-kb dsDNA was first positively supercoiled (by +50 turns) and then RepD (100 nM) was added (down-arrow). The magnetic field was then rotated (by −100 turns) driving the bead and associated DNA molecule toward 50 turns of negative supercoiling. In this example, the DNA molecule was nicked (diagonal arrow) almost as soon as it started to enter the regime of negative supercoiling. Because the DNA had been nicked it could no longer undergo the characteristic length changes associated with supercoiling. Repeated cycles of 50 positive turns of field rotation were then applied to test if the DNA molecule was “supercoilable”. Here, after 54 minutes, the DNA spontaneously religated, and could again be supercoiled. (B) Cumulative frequency plot showing the number of nicked DNA molecules remaining as a function of time (n = 8). The mean time for religation to occur was 1,420 seconds (~24 minutes), giving a rate constant (dotted line) of 5 × 10−4 s−1. The horizontal lines indicate timing uncertainty due to gaps between applications of the +50 turn test protocol (see (A) above). Experiments were at F = 0.4 pN and 23 °C.
