3428 Nucleic Acids Research, 2005, Vol. 33, No. 10 H2B0: 31–124) in each human histone subunit onto their equivalents from other species; thus, an overall root-mean- square deviation (r.m.s.d.) was estimated to be as small as 1.3 s (1.30, 1.29 and 1.41 s for the above NCPs, respect- ively). However, the corresponding DNA duplexes, wrapping the histone octamers, exhibit substantial discrepancies with an r.m.s.d. of 3.0 s (3.04, 2.92 and 2.98 s, respectively), in spite of their completely identical DNA sequences (11,13,14). The detailed structures of the histone–DNA inter- actions are also conserved from yeast to human, including the participation of identical amino acids. Notably, the path of the nucleosomal DNA, coiling around the histone octamer, is dramatically altered in human NCP as compared with those of other organisms. Alteration of the nucleosomal DNA path The two 73 bp DNA halves ( 72 to 0 and 1 to 73, see Figure 2A) of the superhelix in the crystal structure of a human NCP adopted remarkably different structures, with an r.m.s.d. value of 4.19 s (all atoms). This value is sub- stantially larger than those of other NCP structures, such as 2.55 s for Xla-NCP (PDB code 1KX3) (11), 2.44 for yeast NCP (PDB code 1ID3) (14) and 2.67 for chicken NCP (PDB code 1EQZ) (13), demonstrating that the human nucleosomal DNA takes a different path. The superposition of 18 or 19 bp DNA segments between the human and X.laevis structures allowed us to identify the local conformational change in human NCP. As shown in Figure 2A, two segments ( 53 to 18 and 37 to 73, see the legend) present strikingly higher r.m.s.d. values than the others, indicating that the human nucleosomal DNA exhibits a local change in comparison with that of Xla-NCP. The bending behavior of the nucleosomal DNA path in Xla-NCP was successfully evaluated by comparing its base- pair-step parameters (roll, tilt, shift, slide and twist) with the ideal superhelical DNA (16). According to the same comput- ing procedure, we compared the bending behaviors of the nucleosomal DNA path between human NCP and Xla-NCP, and thereby revealed their structural discrepancies at the base pair level. The roll parameter is almost equivalent between two NCPs, although slight differences exist in two segments ( 33 to 21 and 52 to 61), which belong to each region of the altered DNA path (data not shown). The tilt parameter is remarkably different in entire DNA regions (data not shown). The shift, slide and twist parameters of human NCP indicate that two segments ( 53 to 18 and 37 to 73) are quite different from those of Xla-NCP (Figure 2A). Particularly, the twist parameters provide critical informa- tion about the alteration of the supercoiling DNA paths. In human NCP, superhelix axis location (SHL) 5 ( 57 to 46; Figures 2A and 3A, pink ellipsoid and circle) and SHL 5 (46 to 57; Figures 2A and 3B, green ellipsoid and circle) exhibit twists of 9.8 and 9.5 bp per turn, respectively. In contrast, the corresponding values in Xla-NCP are 10.7 and 10.6 bp per turn, respectively. This result indicates that the human DNA path has a much tighter twist than the X.laevis DNA path, and thus the human base pairs are shifted by 1 bp to the 30-side of the I chain (Figure 2B and D). On the other hand, the twist of SHL 2 ( 27 to 16; Figures 2A and 3A, orange ellipsoid and circle) is 10.8 bp per turn, while Xla-NCP shows 9.7 bp per turn. Therefore, in this region, the human DNA path is looser, by 1 bp, in the twist than the X.laevis DNA path (Figure 2C). This indicates that 1 bp shifts, caused by the tighter twist around SHL 5 and SHL 5, were compensated by the looser twist around SHL 2 and 1 bp extension around SHL 7 at the terminus, respectively. These consecutive base pair shifts can essentially account for the high r.m.s.d. values and the parameter differences between human and X.laevis NCPs. Similar differences in the DNA pathway were also observed in comparison with the NCP structures from chicken (13) and yeast (14) (data not shown). The regions around SHL 5, 2 and 5 do not contact the histones, and their conformations appear to be relatively susceptible to the environment. Thus, it is not surprising that the alteration of the human nucleosomal DNA path is concentrated within these regions. However, the B-factors of the DNA backbones at SHL 5 and 5 are lower in human NCP than those of NCPs from other species, including X.laevis (Figure 2A). This suggests that some factors constrain the local alterations in these regions of the human nucleosomal DNA. Conversely, the B-factors at SHL 2 have higher values than the other regions. The 1 bp shift in the two DNA regions (SHL 5 to 2, SHL 5 to 7; black arrows in Figure 3A and B) is clearly illustrated by the best superposition of the histone octameric structures alone between human and X.laevis NCPs. In the altered region (SHL 5 to 2) of human NCP, the phosphate groups, bound to the histones, are also shifted by 1 bp to the 30-side in comparison with that of Xla-NCP (Figures 2A and 3A). However, the interactions of the DNA backbones with the histones are invariant, as revealed from the involvement of the DNA-binding motifs of a1 in H2B, a1 in H2A, loop1 in H2A, loop2 in H2B, loop2 in H4 and loop1 in H3. Similarly, the phosphate groups in the SHL 5 to 7 region move to the 30-side, while maintaining the same binding modes by loop1 in H2B, loop2 in H2A and aN and tail in H3 (Figures 2A and 3B). Figure 2. Comparison of the DNA parameters between human and X.laevis NCPs. (A) B-factors of phosphates, metal binding sites, r.m.s.d. values and twist base- pair-step parameter of the 146 bp DNA compared between human and X.laevis NCPs. The base pair numbers ( 72 to 73) and SHL ( 7 to 7) labels are the same as those in the earlier structures (11,12). The primary bound-phosphate groups are indicated above the base sequence by the pointers showing the strand direction (dark, 30!50; light, 50!30) and the interacting histone motif (L1, loop1; L2, loop 2; A1, a1). B-factors (A˚ 2) are plotted for the 50 phosphate group of each base (brown for the I chain and forest green for the J chain). Mn2+ binding sites are indicated above the base sequence by the yellow and light blue arrows (solid, binding the DNA major groove; broken, binding the DNA minor groove) in the structures of human and X.laevis NCPs (PDB code 1KX3) (11), respectively. The r.m.s.d. values below the base sequence were calculated by the superposition of 18 or 19 bp between two NCPs. The twist base-pair-step parameters were plotted as open circles and closed squares for the human (yellow line) and X.laevis (light blue line) nucleosomal DNA, respectively. The pink, orange and green ellipsoids of the twist parameters represent the corresponding regions illustrated by those in Figures 3A and B and Figure 4A. (B–D) The simplified stereo model of the DNA duplexes in the regions surrounded by the ellipsoids in (A). The differences in helical twist are shown by the superposition between human (yellow) and X.laevis (light blue; PDB code 1KX3) (11) NCPs, based on the histone octamers alone. Numbers presented for each base pair are the same as those in Figure 2A.