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UNWINDING THE DOUBLE HELIX: USING DIFFERENTIAL MECHANICS TO PROBE CONFORMATIONAL CHANGES IN 194 DNA Accuracy of the Calculated Results Once the energetics governing transition under specific environmental conditions have been fit based on the transition behavior of one sequence, the accuracy of the analytical methods can be assessed by comparing their predictions with experimental results on other molecules (Benham, 1992). We did this for six DNA molecules synthesized by David Kowalski, a biochemist studying the role of strand separation in initiating replication (Kowalski and Eddy, 1989). Starting from a parent DNA molecule pORIC, Kowalski made various modifications. pDEL16 has a 16-base pair deletion from the replication origin site of pORIC. pAT105 and pGC91 were made by inserting an A+Târich 105-base pair segment and a G+Cârich 91-base pair segment, respectively, into the deletion site of pDEL16. pAT1051 and pGC911 have the same insertions, but placed in reverse orientation. The complete DNA sequences of these plasmids were provided to the author by Dr. Kowalski (private communication). The transition profiles of these molecules were calculated using the energetics appropriate to the experimental conditions, which were the same as in the pBR322 experiments from which the energy parameters were derived. Figure 7.3 shows the computed transition profiles around the duplex unwinding element (DUE) of the origin site for the four plasmids of greatest interest. The region where strand separation was detected experimentally is shown by a double line in each case. Less separation was detected experimentally at this location in the pORIC plasmid than in the other two transforming molecules, and none was detected in pDEL16 or in the other two molecules whose profiles are not shown in the figure. These experimental results are in close agreement with the present predictions. In fact, the agreement may be even better than the figure indicates. Because the experimental method detects separation only in the interiors of open regions, the actual separated sites are slightly larger than what the experiment detects. These results show that the present methods for analyzing superhelical strand separation are highly accurate. The extensive variations in the locations of separated regions that result from minor sequence alterations are precisely depicted. The relative amounts of transition at each site also agree closely with experiment. The superhelicity required to drive a specific amount of separation is within 7 percent of the observed value, which reflects the limit of accuracy with which extents of transition are