X-Ray Structure of D(GCGAAGC); Switching of Partner for G:A Pair in Duplex Form

X-ray structure of d(GCGAAGC); Switching of partner for G:A pair in duplex form

Tomoko Sunami, Jiro Kondo, Masaru Tsunoda1, Takeshi Sekiguchi1, Ichiro Hirao2-3, Kimitsuna Watanabe4, Kin-ichiro Miura5 and Akio Takenaka Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan, 1College of Science and Engineering, Iwaki Meisei University, Iwaki 970-8551, Japan, 2RIKEN GSC, Wako-shi, Saitama 351-0198, Japan, 3Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904 Japan, 4Graduate School of Engineering, University of Tokyo, Tokyo Downloaded from https://academic.oup.com/nass/article/2/1/181/1058440 by guest on 27 September 2021 113-8656, Japan and 5Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan

ABSTRACT at BL18b of Photon Factory in Tsukuba. Another data set Crystal structure of a DNA fragment d(GCGAAGC), with X=1.00A was collected for structure refinement at 1.6A known to adopt a stable mini-hairpin structure in resolution. Initial phases were successfully estimated with solution, has been determined at 1.6A resolution. Two the program SOLVE. Nine cobalt-hexamine cations in total heptamers are associated to form a duplex with a were located in the unit cell (a=48.7, 6=48.9 and c=63.8A) molecular two-fold symmetry. Three duplexes in the with the space group P212,21. On a density map modified by asymmetric unit have a similar structure. At the both solvent flattening, the molecular structures of six heptamers ends of each duplexes, two Watson-Crick G:C pairs (for three duplexes) in the crystallographic asymmetric unit constitute the stem region. In the central part, two were constructed by tracing their phosphate-ribose sheared pairs of G:A and A:G are formed, the two G backbones with the individual bases. The atomic parameters bases being stacked as well as the two A bases. At this were refined with the program CNS (R=19.5% and point, the two strands are crossed between the two base- *&ee=23.2%). stacked columns. The adenine moiety of the bulged A5 residue, which intercalates between the A4 and G6 residues, makes a small bending of the duplex at the two sites. The difference between the bulge-in structure of d(GCGAAGC) and the zipper-like duplex of d(GCGAAAGC) is ascribed to switching the partner of the sheared G: A pairs.

INTRODUCTION For storage of genetic information, DNA is the highly sophisticated as a chemically stable molecule that has complementarities in a duplex form. However, DNA can have several functions when it exists as a single strand like RNA. Recent in vitro selection make possible to create such functional DNAs. Contrarily the structural basis for designing such molecules is very few. We found that DNA c7' fragments containing a sequence of d(GCGAAAGC) or d(GCGAAGC) exhibit extraordinary properties [1-4]. A Fig. 1. A stereo-pair diagram of a bulge-in duplex mini-hairpin structure was postulated for both fragments structure. The two heptamers of d(GCGAAGC) are associated with each other including two bulge-in A from NMR and CD experiments [2,4]. But they show the residues and two sheared G:A pairs. structural versatility that the octamer with the sequence d(GCGAAAGC) adopts a zipper-like duplex structure in crystalline state [5]. For more extensive study we started X- RESULTS AND DISCUSSION ray analysis of the heptamer with d(GCGAAGC). The crystallographic asymmetric unit contains six heptamers, two of which form a duplex in crystalline state. The three EXPERIMENTAL duplexes have a similar structure within the r.m.s.d. of 0.7A, A DNA heptamer with the sequence d(GCGAAGC) was further each having a molecular two-fold symmetry, as synthesized and crystallized by the hanging-drop vapor shown in Fig. 1. At the both ends of the duplex, the two diffusion method. Suitable crystals for X-ray experiment consecutive G:C pairs constitute the stems (G^C/, C2:G6\ were obtained from cobalt-hexamine containing solutions. G6:C2*, and C7:G,*, where asterisks specify the counter 2.0A Resolution data for MAD phasing were collected with strand). In the central part, two sheared G:A pairs are synchrotron radiation (X=1.00, 1.6046, 1.6053 and 1.6090A) formed between G3 and A4* and between A4 and G3* through 182 Nucleic Acids Research Supplement No. 2 the N3(G)...N6-H(A*) and N2-H(G)...N7(A*) hydrogen bonds. For this consecutive sheared G:A and A:G pairs, the two guanine bases are stacked, as well as the two adenine bases are stacked, as shown in Fig. 2. Therefore, their helical twist angle becomes larger (~90°). At the result, the successively stacked columns of bases are crossed between the two strands so that the column GjC2G3 is continued to another column G3*C2*G|*, and the column C7*G6*A5'A4* is continued to the column A4A5G6C7. The Watson-Crick sites of the four residues G3, A4, G3* and A4* are exposed, as well as the major groove sites of G3 and G3*, and the minor Downloaded from https://academic.oup.com/nass/article/2/1/181/1058440 by guest on 27 September 2021 groove sites of A4 and A4*. These structural features of G:AxA:G crossing are similar to those essential in hammerhead ribozymes [6,7].

Fig. 4. A superimposition of the stem parts between the bulge-in duplex (black line) and the zipper-like duplex (gray line) structures. The G3 base plane swings (arrows with broken lines) to switch the pairing partner from A5* to A4*, accompanying a large movement of A4* residue.

It is interesting to compare the present bulge-in duplex structure with that of the zipper-like duplex of d(GCGAAAGC) [5]. Addition of an adenosine residue in the central part changes the partner of the G:A pair, as shown in Fig. 4. In the zipper-like duplex (refer the residue numbering in Fig. 3(b)), the pairing occurs between the G3 Fig. 2. The stacked two sheared G:A pairs at the and A5* residues and the G3 and A5* pair is stacked on the G:AxA:G crossing. C2:G6* pair. A superimposition between the two structures at the stem parts (G,:C7* and C2:G6* pairs) suggests that an angular movement (swinging) of the G3 base plane between the A4* and A5* bases is a determinant for selecting the type of the duplex structures. Switching the partner from A4* to A5* (bulge-in to zipper-like) induces a large movement of the A4" residue. In the case of the zipper-like duplex, the A3-5 residue is stacked on the G3 residue. Therefore, in the zipper-like structure, the G:AxA:G crossing is not formed, instead the four central adenosine residues are stranded by only stacking, their bases being largely exposed [5].

REFERENCES 1. Hirao, I., Naraoka, T., Kanamori, S., Nakamura, M. and Miura, K. (1988) Biochem. Int., 16, 157-162. 2. Hirao, I., Nishimura, Y., Naraoka, T., Watanabe, K., Arata, Y. and Miura, K. (1989) Nucleic Acids Res., 17, 2223-2231. 3. Hirao, I., Nishimura, Y., Tagawa, Y., Watanabe, K. and Fig. 3. The secondary structures of the bulge-in duplex Miura, K. (1992) Nucleic Acids Res., 20, 3891-3896. (a) and zipper-like duplex (b) found in crystal structures 4. Yoshizawa, S., Kawai, G., Watanabe, K., Miura, K. and of d(GCGAAGC) and d(GCGAAAGC). Hirao, I. (1997) Biochemistry, 36,4761-7. Another novel feature is that the adenine moieties of 5. Sunami, T., Kondo, J., Chatake, T., Hirao, I., Watanabe, K., Miura, K. and Takenaka, T. (2001) Nucleic Acids Res., bulged A5 residue stay inside the duplex, intercalating between the A4 and G6 residues to form a bulge-in duplex. Supplement 1,191-192. On the counter strand, however, the C2* and G3* bases are 6. Pley, H.W., Flaherty, K.M. and McKay, D.B. (1994) still stacked with a small inclination. Thus the stem helix is Nature, 372,68-74. slightly curved by this bulge-in residue. The similar 7. Scott, W.G., Finch, J.T. and Klug, A. (1995) Cell, 81, situation occurs at the A5* residue, so that the two curved 991-1002. stems make a smooth bending of the duplex.