same analysis to DNAs with hyperperiodic sequences. Plots of mean squared end-to-end distances of hyperperiodic DNA samples, PPC (Figure 4a, colored symbols) and SQ322 (Figure 4b, red circles), showed that AA/TT period- icity induces extra bending in the DNA structure. This was demonstrated by signiﬁcant deviations of the hyperperiodic DNA from the predictions of the WLC model using a persist- ence length of 50 nm. Subsequent analysis to determine the source of this devi- ation took into account distributions of absolute values of cur- vatures j C j . As a check for the validity of this analysis, we found that curvature distributions had a maximum at zero, showing equal probability for the observation of positive and negative curvatures. The value sC (Table 2) obtained from Gaussian ﬁts to the jCj distributions indicated how often large curvatures were present in the trace or the degree of bendability. Figure 4c and d showed that hyperperiodic DNA (PPC and SQ322) accommodates high curvatures more readily than non-periodic DNA (Control and SQ100). However, it is not possible from the value of sC alone to attri- bute the increased presence of high curvatures to increased ﬂexibility or to the presence of intrinsic bends: indeed either intrinsic curvature or unusual ﬂexibility will widen the C distribution (Supplementary Data). Nevertheless, it is accur- ate to say that if the DNA does not have an intrinsic bend, then the value of sC directly reﬂects the degree of ﬂexibility. One characteristic feature of the C.elegans hyperperiodic sequences analyzed in this work is the distance over which substantial periodicity is maintained. In the AFM images, this corresponds to what appears to be a considerable distance over which the handedness of the curvature persists (Figure 3a). We quantiﬁed the distance D over which handed- ness was correlated using Equation 2. These correlation plots were computed for the PPC and the Control DNA (Figure 5a). It was observed that at a given distance D the probability of ﬁnding a curvature with the same sign was signiﬁcantly higher in the hyperperiodic DNA than in non-periodic DNA. The curvature correlation increased with larger values of L. This indicates that AA/TT periodicity induces a long- scale effect in the structure of DNA and provides a strong argument for bending as opposed to ﬂexibility, since ﬂexibil- ity would deﬁnitely not increase the correlation distance. In principle, the process of adsorption itself could cause switch- ing of handedness as the DNA might ﬂip over at certain points, leading to an underestimation of the correlation distance D, compared to the 3D structure in solution. A complementary approach to analyze the presence or absence of long-scale correlated intrinsic bends consists of calculating the mean curvatures per molecule (Figure 5b and c). This analysis showed a maximum at zero for the non-periodic DNA samples (control and SQ100), in agree- ment with purely random bending. A clear peak, however, was found for both of the hyperperiodic DNA samples (PPC and SQ322), suggesting the presence of a large-scale intrinsic bend with an average curvature of 0.01 nm 1 (at L ¼ 5 nm). It should be noted that we underestimate the bending of the DNA, because it is not clear how the bent regions absorb to the mica, resulting in a mixture of regions with positive and negative curvature. The absence of intrinsic bending for the non-periodic SQ100 DNA suggests that its widened curvature distribution (Figure 4d) could be due to Nucleic Acids Research, 2006, Vol. 34, No. 10 3065 an increase in ﬂexibility compared to the non-periodic Con- trol (Figure 4c). For hyperperiodic DNA, however, it was not possible to address ﬂexibility, as ﬂexibility is convoluted by intrinsic bending in the curvature distributions. However, the variety of shapes displayed in the AFM images (Figure 3d) indicated that hyperperiodic DNA seems to be ﬂexible enough to adopt straight and strongly bent conforma- tions. DNA sequences from numerous organisms have been shown to contain regions with periodic character correspond- ing to the helical repeat of 10–10.5 bp. Despite a number of cases in which structural roles have been assigned to relat- ively short periodic regions (17), the functions of extended periodic regions in DNA are unknown. Of the numerous ‘model’ genomes that have been subject to intensive analysis, C.elegans has (at least by some measures) the strongest peri- odicity signal (19,24). Given that 1–2% of the base pairs in the C.elegans genome appear constrained by their contribu- tion to periodic character (19), it seems likely that these sequences have substantial roles in the evolution and/or func- tion of the genome. One role that has been considered by vir- tually every investigator who has studied these sequences has been to constrain or otherwise inﬂuence nucleosome position- ing. Despite these proposals, it remains a possibility that other extended surfaces in the nucleus are implicated in the observed long-range sequence relationships. In summary, we analyzed molecules with a greater degree and extent of periodicity than any previously analyzed DNA. We used gel electrophoresis and AFM to investigate the effect of long PATCs in the structure of the genome of the nematode C.elegans. Gel electrophoresis showed that hyper- periodic DNA migrates more slowly than non-periodic DNA. We quantiﬁed trajectories of equilibrated molecules on a mica surface obtained with high-resolution AFM, and proved that the PATCs exhibit large-scale intrinsic bending. This explained the anomalous gel migration. Finally, we studied two nucleosome-length DNA fragments that survived vigor- ous Micrococcal nuclease digestion in situ, and likewise found the presence of an intrinsic bend for the fragment con- taining hyperperiodic sequences. The fragment without intrinsic bending exhibited increased ﬂexibility. This is con- sistent with the idea that bendability of the DNA might be a relevant factor in promoting binding to nucleosomes. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We are grateful to Jamie Fleenor and Chaya Krishna for expert technical assistance, and to Javier Lopez-Molina and Michael McCaffery for early advice on protocols. We thank Fabian Czerwinski for the preliminary AFM experiments. F.M.-H. is supported by a postdoctoral contract from the Dutch research program NanoNed. S.M.J. is supported by a postdoctoral fellowship PF-05-121-01-DDC from the American Cancer Society. The laboratory of N.H.D. acknowl- edges financial support from FOM (Dutch Foundation for Fundamental Research on Matter) and NWO (Netherlands