3064 Nucleic Acids Research, 2006, Vol. 34, No. 10 Figure 5. Measurement of the intrinsic curvature in hyperperiodic C.elegans DNA. (a) Curvature correlation function (Equation 2 in the main text) for PPC and Control DNA calculated for curvatures measured at contour length separations of L ¼ 5, 15 and 25 nm. A long-range persistence of handedness in hyperperiodic DNA is evident from the graph, indicating the presence of intrinsic bending. (b) Normalized histograms of absolute mean curvatures per molecule |<C>molecule| for the PPC and the control DNA sample for L ¼ 5 nm and for L ¼ 25 nm [inset in (b)]. (c) |<C>molecule| histograms for SQ100 and SQ322 for L ¼ 5 nm. Hyperperiodic DNA showed a preferred mean curvature per molecule of 0.01 nm 1 at L ¼ 5 nm, consistent with the presence of intrinsic bending. Bins and associated errors (square root of number of points in the bin) were normalized by the area of the histogram. D. Qualitatively similar correlation functions were predicted from theoretical calculations for chains with long-range cor- relations (28). Therefore, these data are strongly indicative of the presence of an intrinsic bend rather than increased ﬂexibility. A complementary approach to the correlation distance con- sisted of calculating an average curvature per molecule and taking its absolute value j<C>moleculej. The distributions of absolute curvature per molecule are shown in Figure 5b and c. The statistics in these experiments is poorer (resulting in lar- ger error bars) than in the j C j distributions (shown in Figure 4c and d), as per deﬁnition the total number of values for the histogram equals the number of molecules imaged. Figure 5b shows j<C>moleculej distributions for the PPC and the non-periodic control DNA, and Figure 5c shows j<C>moleculej for the SQ100 and SQ322 fragments. Distribu- tions were calculated using a spacing of L ¼ 5 nm to calculate the curvature values, except the inset in Figure 5b, which used L ¼ 25 nm. The distributions were ﬁt with Gaussian functions. As expected, the distributions for both non- periodic DNA molecules had a maximum at curvature zero. However, the distributions for the two hyperperiodic DNA samples displayed an offset, j<C>0,molecule L¼5 nmj of 0.01 nm 1. This implies that a given molecule has the tend- ency to bend either more towards the right than towards the left or vice versa. This observation can only be explained by the presence of an intrinsic curvature, as the distributions for the ‘random’ control DNAs had a maximum at around zero. We note that the effect is more pronounced for the long DNA samples, as here more curvature values contribute to the mean curvature, which can directly be seen from the width of the distributions s<C>molecule (Table 2). Similar conclusions can be drawn from the analysis done at L ¼ 25 nm (inset in Figure 5b), showing that the detection of an intrinsic bend is independent of the spacing L used. DISCUSSION We report anomalous gel migration of several fragments of the nematode C.elegans genome with unusual periodicity of AA/TT dinucleotides. AFM allowed detailed characterization of the bending of these DNA structures showing that PATCs generate intrinsic bending. Despite the presence of an intrinsic bend, the AFM images also showed that hyperperi- odic DNA can still adopt a wide range of bending conﬁgura- tions (Figure 3d), suggesting that ﬂexibility is retained. In order to be able to extract quantitative information related to bending of DNA, we ﬁrst veriﬁed that our DNA contours reﬂect 2D equilibrium states and that the average behavior can be accurately described by the WLC model. For this, we used as a control the plasmid pGem-3Z, which had no unusual periodic character. We observed that the aver- age trajectory of adsorbed control DNA molecules fulﬁlled several criteria: (i) the distribution of curvatures was Gaus- sian (Figure 4c, black squares), and (ii) the mean square end-to-end distance followed Equation 1 with the accepted value for the persistence length (Figure 4a, black squares). Thus, we conclude that the WLC describes accurately the average trajectory of our molecules and that these trajectories reﬂect equilibrium states of the molecules on the mica sur- face. The fact that the control DNA molecules are equilib- rated on the surface is crucial for allowing us to extend the