3062 Nucleic Acids Research, 2006, Vol. 34, No. 10 Figure 4. Analysis of unusual character in hyperperiodic DNA from C.elegans using AFM. (a and b) Plots of <R2> versus contour length spacing L of the ends of the PPC, control DNA, SQ100 and SQ322 samples. Different sets of data in (a) correspond to different experiments, mica surfaces and sample preparations. In total, 190 PPC fragments were studied. The control DNA follows the prediction given by the worm-like-chain model with a persistence length of 51 nm. The hyperperiodic DNA regions from the C.elegans genome clearly show a very different behavior, characterized by lower <R2>. To guide the eye, predictions from WLC with different values for the persistence length are included (solid grey). The five curves included are for P ¼ 20, 30, 40, 50 and 60 nm. The slope of the curves increases as P increases. Bending of the DNA, is evidenced by lower <R2> than predicted by WLC. (c) Normalized histograms of absolute curvature |C| for the PPC and the control DNA sample for L ¼ 5 nm and for L ¼ 25 nm [inset in (c)]. (d) Normalized absolute curvature |C| histograms for SQ100 and SQ322 for L ¼ 5 nm. Distributions were fit to a Gaussian function centered at zero. The hyperperiodic DNA consistently showed larger standard deviations, independently of the spacing L [inset in (c)]. Bins and associated errors (square root of number of points in the bin) were normalized by the area of the histogram. We note that occasionally one cannot distinguish the error bars because they are smaller than the points. sequence periodicity. As described by Rivetti et al. (26) DNA equilibrates on a mica surface when adsorption is promoted by divalent ions such as Mg2+. The behavior of DNA molecules under these conditions can be predicted by the WLC model (26). The WLC describes a mean trajectory of the polymer on the mica with the following equation: hRs‚ 2 sþLi ¼ 4P L þ 2Pðe L/ð2PÞ 1Þ ‚ 1 where P is the persistence length of the polymer, L is the con- 2 tour length spacing and hRs‚ sþLi is the mean-squared separa- tion between points along the chains located at distances s and s+L from the end of the molecule. The average is com- puted over s and over all observed contours. The averaging is done up to the upper limit in the contour length of the molecule L0 (for the PPC, L0 equals 325 nm) minus the con- tour length spacing L analyzed, thus 0 < s < L0 L. Using this analysis, the control DNA and the non-periodic short fragment SQ100 displayed exactly the behavior pre- dicted by the WLC model; the hyperperiodic DNA, however, did not. Figure 4a shows hRs2‚ sþLiversus L for the Control 2 and PPC samples. We calculated hRs‚ sþLi up to a contour length spacing of L ¼ 200 nm. This is because the length of the segment studied in the PPC was 325 nm, necessarily restricting one to values of L < L0 to obtain a statistically reliable calculation for all L. For consistency, we also used an upper limit for L of 200 nm for the control DNA. A ﬁt using Equation 1 was done for the control DNA (Figure 4a, black line). The ﬁt yielded a value for the persistence length of 51 nm, in agreement with published results (26,27). These data were consistent with the hypothesis that our sample pre- paration yields contours that reﬂect equilibrium 2D chain conformations. We repeated the same type of analysis on the PPC, tracing 325 nm (953 bp) from both ends. The use of a palindrome construct is very convenient because it avoids ambiguity in the sequence analyzed: the traced fragments always included the hyperperiodic Fragment W4 from C.elegans independently of which end was selected. In this way, 190 fragments coming from three sets of experiments done on different days, mica surfaces and preparations were traced and analyzed. Remarkably, the same analysis done on 2 the PPC showed signiﬁcantly smaller hRs‚ sþLi than predicted by the WLC model (Figure 4a), which is indicative of extra bending. To guide the eye we included plots of Equation 1 using P ¼ 20, 30, 40, 50 and 60 nm (solid lines in Figure 4a). The PPC data curves mostly overlaid the curve of P ¼ 30 nm. It should be noted, though, that the WLC model does not necessarily hold for intrinsically curved DNA. We conducted a similar analysis using samples SQ100 and SQ322. In this case due to the shorter length of the 2 molecules we plotted hRs‚ sþLi up to a contour length spacing of L ¼ 47.5 nm (Figure 4b). This required tracing many more molecules to have comparable statistics with those for the PPC sample. We traced 692 and 653 molecules for SQ100 and SQ322, respectively. The analysis showed that SQ322