A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 å resolution

Research Article

849

A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 Å resolution William Shepard1*, William BT Cruse2, Roger Fourme1, Eric de la Fortelle3 and Thierry Prangé1,2 Background: The replication origin of the single-stranded (ss)DNA bacteriophage G4 has been proposed to fold into a hairpin loop containing the sequence GCGAAAGC. This sequence comprises a purine-rich motif (GAAA), which also occurs in conserved repetitive sequences of centromeric DNA. ssDNA analogues of these sequences often show exceptional stability which is associated with hairpin loops or unusual duplexes, and may be important in DNA replication and centromere function. Nuclear magnetic resonance (NMR) studies indicate that the GCGAAAGC sequence forms a hairpin loop in solution, while centromere-like repeats dimerise into unusual duplexes. The factors stabilising these unusual secondary structure elements in ssDNA, however, are poorly understood. Results: The nonamer d(GCGAAAGCT) was crystallised as a bromocytosine derivative in the presence of cobalt hexammine. The crystal structure, solved by the multiple wavelength anomalous dispersion (MAD) method at the bromine K-edge, reveals an unexpected zipper-like motif in the middle of a standard B-DNA duplex. Four central adenines, flanked by two sheared G·A mismatches, are intercalated and stacked on top of each other without any interstrand Watson–Crick base pairing. The cobalt hexammine cation appears to participate only in crystal cohesion.

Addresses: 1LURE, Bâtiment 209d, Université Paris-Sud, 91405-Orsay Cedex, France, 2Chimie Structurale Biomoléculaire (URA 1430 CNRS), 93017-Bobigny Cedex, France and 3Laboratory of Molecular Biology, MRC, Hills Road, Cambridge CB2 2QH, UK. *Corresponding author. E-mail: [email protected] Key words: adenine tract, G·A mismatch, hairpin, MAD, single-stranded DNA, zipper-like DNA Received: 6 April 1998 Revisions requested: 6 May 1998 Revisions received: 21 May 1998 Accepted: 21 May 1998 Structure 15 July 1998, 6:849–861 http://biomednet.com/elecref/0969212600600849 © Current Biology Ltd ISSN 0969-2126

Conclusions: The GAAA consensus sequence can dimerise into a stable zipper-like duplex as well as forming a hairpin loop. The arrangement closes the minor groove and exposes the intercalated, unpaired, adenines to the solvent and DNA-binding proteins. Such a motif, which can transform into a hairpin, should be considered as a structural option in modelling DNA and as a potential binding site, where it could have a role in DNA replication, nuclease resistance, ssDNA genome packaging and centromere function.

Introduction On the basis of their conserved nucleotide sequence, the secondary structures of single-stranded (ss)DNA at the replication origins of bacteriophages and parvoviruses have been proposed to form hairpin structures [1–4]. In bacteriophage G4, the starting point of negative-strand synthesis, initiated by the Escherichia coli dnaG (primase) protein, lies just before one of three regions proposed to form hairpin loops [3]. The proposed hairpin at this site comprises an 8 base-pair (bp) stem and the loop sequence GAAAGCC. Studies on synthetic analogues of this hairpin revealed that fragments containing a GAAA consensus are just as stable as the original proposed hairpin [5]. These fragments exhibit very high melting temperatures (Tm > 75°C) [5], resistance to nucleases [6] and fast gel mobilities [7], even with only two Watson– Crick base pairs in the stem. On the basis of preliminary NMR studies and molecular dynamic calculations, a hairpin structure with a minimal sequence of GCGAAAGC

was proposed to account for these properties [8–10]. Interestingly, the d(GCGAAAGC) fragment is much more stable than its RNA analogue [9], which is a member of the r(GNRA) family of RNA tetraloop sequences (where N = A, C, G or U and R = A or G) and are well documented for their stability [11–14]. It is interesting to note that the loop sequence d(GAAA) is similar to the highly conserved repetitive DNA sequence (GGAAT)n found in human centromeres [15], and occurs with moderate frequency in fission yeast tandem repeats dg and dh [16,17]. The sequence is also found at the core of the 17 bp repeat consensus that associates with the centromere-binding protein known as CENP-B [18–20]. In its single-stranded form the (GGAAT)n repeat sequence, as well as its (GNAAT)n variant, shows thermal stabilities comparable to Watson–Crick duplexes formed between complementary strands [15]. These stable single-stranded tandem array structures were originally proposed to form a

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duplex involving mismatched G·A base pairing [15]. Later, however, NMR studies on analogues of this centromerelike repeat were shown to form a variety of stem loop and duplex structures [21–24]. Here we present the crystal structure of the d(GCGAAAGCT) sequence at 2.1 Å resolution, which has been solved by the multiple wavelength anomalous dispersion (MAD) method after incorporation of a bromine onto the second cytosine. The sequence folds into a self-forming duplex structure containing a novel adenine-rich zipper-like motif embedded in standard B DNA. None of the hairpin structures as proposed from NMR studies was observed [8–10]. This novel structure demonstrates how DNA can overcome a noncomplementary sequence to fold into a duplex, and raises questions about secondary structure in ssDNA systems.

RNA oligomers [29]. In the present deoxy structure, the G·A mismatches occur after only two Watson–Crick base pairs, suggesting that this type of mismatch is a rather stable configuration. The substantially buckled nature of Figure 1 (a)

Results Description of the structure

The structure of d(GCGAAAGCT) is an elongated and stretched duplex with a four-step zipper-like motif at the adenine core. The asymmetric unit contains only one DNA strand, but self-association about a twofold crystallographic axis generates a duplex structure (Figures 1 and 2). Although the duplex at both ends folds into standard B DNA with the formation of two classic Watson–Crick base pairs G(1)–C(8*) and C(2)–G(7*) (where the asterisk denotes a base from the crystallographically related molecule), the rest of the organisation is completely different. Two uncommon, buckled G·A mismatches occur at steps 3 and 6, and the core adenines are unpaired and intercalated into a zipper-like arrangement. This motif has the effect of unwinding the helix, leading to the elongation of the duplex. As the base rise still remains at normal B DNA stacking distances (an average value of 3.3 Å), an elongation of the phosphodiester backbone accommodates the deformation induced by the two consecutive intercalations. This type of deformation of the phosphodiester backbone is similar to those observed in DNA–drug complexes [25,26]. The intercalated adenines are bracketed on either side by two G·A mismatches. As illustrated in Figure 3, the central adenines are aligned in such a fashion that they stack between the guanines of the G·A mismatches, and thus form a well aligned polypurine stack of six bases. Table 1 highlights the dramatic conformational differences in the phosphate backbone compared to the more standard structures of DNA. The third and sixth steps of the duplex are G(anti)·A(anti) mismatches which are characterised by the formation of two hydrogen bonds at G(N3)–A(N6) and G(N2)–A(N7). This type of G·A mismatch, referred to as a sheared base pair, has been observed in DNA oligomers (for a review see [27] ), as well as at a divalent ion binding site in domain II of the hammerhead ribozyme [28] and in unusual

(b) Base spacings

T9* G1

n

C2

n

G3

n

C8* G7* A6*

Watson–Crick pairs G•A mismatch

A4 A5*

2n

Adenine zipper

A5 A4*

2n A6 n

G7

n

C8

T9

G3* C2* G1*

G•A mismatch Watson–Crick pairs

Structure

The zipper-like motif in DNA. (a) The initial experimental electrondensity map at 2.35 Å resolution with the final model superimposed. Contouring is at 2σ above the mean density level. The region shown is the central part of the intercalating adenines. The symmetry-related molecule (in red) is shown to illustrate how the zipper-like dimer is built. (b) Schematic diagram of the base-stacking arrangement in the extended/stretched duplex (the helix is represented unwound). The two ends comprise normal Watson–Crick pairings but the middle of the helix shows the disymmetric extension and alternate intercalations of unpaired adenines with an interspacing parameter n = 3.3 Å. Adenines are shown in blue, guanines in green, cytosines in red and thymines in magenta; the bromine atoms are shown as yellow spheres.

Research Article Structure of an adenine zipper in DNA Shepard et al.

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Figure 2 Comparison of the zipper-like motif to standard B DNA. (a) The zipper-like motif alongside a segment of (b) B DNA. Both structures correspond to an 8 bp stretch (the ninth thymine residue in the zipper-like motif is flipped out of the helix). The zipper-like motif displays the characteristic shape of an X with an elongated phosphodiester backbone. All the adenine bases point in the same direction as the minor groove collapses in the central region. The standard B-DNA structure is more regular and compact.

the G·A mismatches brings the phosphodiester chains together, merging the opposing DNA strands into the zipper-like motif. A severe torsion (kink) is introduced in the helical structure at the G·A mismatches (visible in Figure 3), but this has no effect on the adenine tract itself, which shows a rather straight alignment in the core region (Figures 2 and 3). Other variations of deoxy G·A mismatches are known from X-ray crystal structures and NMR

studies [27,30], but in general they have a destabilising effect compared with Watson–Crick base pairs [31]. Crystals of the zipper-like duplex form only in the presence of metal hexammine salts (cobalt, rhodium or iridium), and a cobalt hexammine cation is clearly apparent in the crystal structure. A dramatic increase was found both in the resolution of the diffraction pattern

Figure 3 Stereo view of the zipper-like motif. A closeup view of the central adenine tract cut out from the duplex. The figure illustrates the substantial buckling of the sheared G·A mismatch located at the bottom of the figure. Atoms are shown in standard colours.

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Table 1 Geometry parameters in the monomer and in the duplex. Intrastrand†

Interstrand‡

Sugar puckerings§

Pn–Pn+1 distances

Step

η

P–P* distances¶

C1′–C1′ distances¶

φ

P

τm

(Å)

#

(°)

(Å)

(Å)

(°)

(°)

(°)

1 2 3 4 5 6 7 8

9.4 9.8 17.4 12.6 –9.6 27.8 13.8 9.5

165 120 160 35 220 205 100 125

33 44 43 35 37 37 39 28

P2–P3 P3–P4 P4–P5 P5–P6 P6–P7 P7–P8 P8–P9

6.60 7.11 6.14 6.29 6.40 6.78 6.27

P2–P8* P3–P7* P4–P6* P5–P5* P6–P4* P7–P3* P8–P2*

18.43 16.29 10.25 9.10 10.25 16.29 18.43

G1–C8* C2–G7* G3–A6* A4–A5* A5–A4* A5–G3* G7–C2* C8–G1*

10.59 10.48 8.0 4.5 4.5 8.01 10.48 10.59

11.5 6.5 11.7 –86 94 –11.7 –6.5 –11.5

G1 C2 G3 A4 A5 A6 G7 C8

†For

comparison, the average values for phosphate–phosphate distances (Pn–Pn+1) within a strand are 5.9, 7.0 and 6.5 Å for A DNA, B DNA and tetraplexes, respectively. Except for the abrupt change at the G·A mismatches, these values are similar to the tetraplex geometry. The inclination angle, η, represents the angle between the mean plane of the individual base and the perpendicular to the helix axis (positive values denote downward inclinations). ‡Interstrand P distances represent the variable diameter of the duplex. Typical values are remarkably constant in A and B DNA (17.55 and 17.75 Å). The adenine zipper squeezes the strands together which dramatically reduces the interstrand P distances in the middle. The line from P5 to P5* is not perpendicular to the helix axis and does not represent the shortest interstrand distance which is P6–P5* (6.36 Å). The C1′–C1′

vectors are usually perpendicular to the helix axis in ‘standard’ B DNA (φ close to 0°). This is true for the three first base pairs, where distances correspond to the canonical B DNA, but not for the others. The inner vectors A4–A5* (and its symmetric A5–A4*), are now nearly parallel to the helix axis, as these adenines are not paired but only stacked. For comparison, the average C1′–C1′ distances are 10.5 Å in both A and B forms of DNA and 9.4 Å in the tetraplex structures. §P and τ are the pseudo-rotation parameters of the sugar puckerings. m These parameters were not constrained during refinement of the structure. Standard deviations are in the range of 5°. The sugars all adopt the ‘southern’ conformation, with the exception of sugar 4 which adopts the ‘northern’ conformation. ¶The asterisk denotes a symmetryrelated atom (second strand of the duplex).

and the mechanical resistance of the crystals, when changing the complexed metal hexammine from cobalt to either rhodium or iridium hexammine — a feature already observed in RNA oligomers [32]. The cobalt hexammine forms strong and specific hydrogen bonds at the G(3) base level. The side-by-side arrangement of the bases of the sheared G·A mismatch turns the N7 and O6 atoms of G(3) outwards from the major groove and allows for formation of hydrogen-bonding interactions with the cobalt hexammine cation. This cation sits on a threefold crystallographic axis and bridges three duplexes at the sheared G·A mismatch (Figure 4). It also occupies a position in one of the solvent channels and makes no interactions to any phosphate oxygens (Figures 4a and 5a). As such, the cobalt hexammine is considered to participate largely in crystal cohesion rather than in duplex stability. The central unpaired adenines are also located about the threefold axis and are retained in a weak triplex association by hydrogen bonds, mediated by a water molecule which is also visible in the initial experimental MAD electron-density map.

considered an important stabilising effect in the crystal formation between two aligned symmetry-related helices. The G–C pairs at both ends are parallel and oriented so that the guanine G(1) is stacked over the symmetry-related cytosine C(8*) and vice versa. The last thymine residue at position 9 was initially introduced in the synthesis in order to promote an intermolecular A–T pair formation as a means of crystal stabilisation. This residue projects out of the helix, however, into a cavity delimited by the packing. The residue is strongly disordered, unpaired and autonomous of the stacking — showing up only as a faint signature in the electron density. In the packing structure, the helices are arranged in such a fashion that a large solvent channel, about 18 Å in diameter, is built about the sixfold axes and oriented along the z axis (Figure 5).

As the double helix runs parallel to the z axis of the crystal lattice, the packing of symmetry-related duplexes on top of each other leads to the formation of continuous doublehelical stacks in the crystal (Figure 5). This style of packing is rather well documented in A and B nucleic acid structures, in both the free and intercalated states [26,32], and is

Discussion Comparison with [GGA]2 motifs and other intercalated structures

The zipper-like motif has certain similarities with three centromere-like DNA oligonucleotides, which form selfassociating duplexes or hairpin structures, as determined from high resolution NMR studies [22,23,33]. In all cases, the oligomers form B DNA family duplexes with two intercalated and unpaired guanine residues bracketed by sheared G·A mismatches, and dubbed the [GGA]2 motif (unpaired and intercalated bases are highlighted in bold). This motif is of particular interest because human

Research Article Structure of an adenine zipper in DNA Shepard et al.

centromeric DNA was discovered to contain d(GGAAT)n tandem repeats, which are just as thermally stable alone as with their complementary strand in Watson–Crick duplexes [15]. Both [d(TGGAATGGAA)]2 [22] and [d(GTGGAATGGAAC)]2 [23] oligonucleotides are noncomplementary sequences that form duplexes with two [GGA]2 motifs. The sequence d(GTGGAATGCAATGGAAC) [33] forms a ‘fold-back’ structure with a GCA hairpin loop at the central cytosine residue (underlined) and the remaining nucleotides folding into a duplex with two (GGA)2 motifs. All of the duplex sugar residues are in the C2′-endo conformation except for the unpaired guanosines which are in the unusual C3′-endo conformation. The remarkable stability of these duplexes, despite having as few as only two Watson– Crick base pairs out of ten, has been attributed to three factors: the interstrand stacking of the unpaired guanines with the guanines in the adjacent G·A mismatch; packing of the sugar residue of the unpaired guanine in between two of the bases of the G·A mismatch; and a hydrogen bond between the unpaired guanine NH2 and a phosphate oxygen from the opposite strand. The zipper-like motif in the [d(GCGAAAGCT)]2 duplex shows base stacking more extended than the [GGA]2 motif. The central adenines stack in between the guanine bases of the two sheared G·A pairs and beyond to extend throughout the whole duplex. Figure 3 illustrates this and how the stacking crosses over to the opposite strand (i.e. the stacking follows the sequence GCGA*AA*AG*C*G*, where bases of one of the strands are denoted with asterisks). As in the [GGA]2 motif, the sugar residue of the first unpaired base (A4 in the present structure) following the sheared G·A mismatch conforms to C3′-endo or north configuration and packs against the adenine of the G·A mismatch. The adenine A4 does not, however, form a hydrogen bond to the phosphate oxygen from the opposite strand as seen for the [GGA]2 motif, because adenine lacks the amino group (NH2) on the C2 carbon atom, which is present for guanine. Interestingly, both the phosphate and adenine in question are in suitable positions to form such a hydrogen bond if the amine group were added. This implies that this hydrogen bond in the [GGA]2 motif is not essential to the formation of the duplex, but does contribute to its overall stability (see below). The sugar residue of the second unpaired and intercalated adenine (A5), reverts back to the C2′-endo or south configuration. This adenine also does not form any other hydrogen bonds to phosphate groups or bases. A consequence of the zipper-like motif is a subsequent pinching together of the phosphodiester chains to bring the interstrand P–P distances between P5 and P6 to within 6.6 Å. This is more marked in the [GGA]2 motif, where the shortest interstrand P–P distance is 8.5 Å [23]. The negatively charged phosphate groups compensate for this by pointing their oxygens in opposite directions and away from each other (see Figure 3). In fact, the

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Figure 4 (a)

W13

W12

G3

A6*

(b)

N7* O6 N4

O6*

N3

N6 N7

N5 N1

N2 N7# O6#

Structure

The cobalt hexammine site. Two views of the cobalt hexammine interactions at the G3 base level, which illustrate the special role of this residue. (a) The 2Fo–Fc map at 2.1 Å resolution of the Co(NH3)63+ and the surrounding guanine residues (contoured at 2σ above the mean density with phases from the refined model). (b) Diagram of the extensive hydrogen-bond network linking the Co(NH3)63+ cation around the threefold crystallographic axis to the N7 and O6 atoms of G3.

conformation of the phosphodiester backbone in the zipper-like motif is analogous to those found in the intercalated ‘i-motifs’ of cytosine-rich DNA oligonucleotides [34–45]. The phosphodiester chains in i-motifs are packed side-by-side in an antiparallel fashion with the sugar residues in C3′-endo or C4′-exo conformations. With the minor groove effectively closed, the bases are turned out

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Figure 5 (a)

(b)

Structure

Crystal packing. (a) Packing of the structure viewed along the z axis showing the large solvent channel located on the sixfold axes (diameter approximately 18 Å). The cobalt hexammine cations lie in narrower channels which also run parallel to the z axis and are located about the threefold axes. (b) Two perpendicular views of the end-to-end packing

of duplexes in the crystal structure that generates a continuous helix along the z axis. The asymmetric unit is shown in yellow, its ‘complementary’ strand in red and the stacked symmetry-related helices in green. This duplex is flattened into a ribbon-like structure compared with the standard B DNA double helix.

to present their Watson–Crick faces along the opposite side of the duplex with respect to the phosphodiester backbone, as is seen for the adenines in the zipper-like motif. However, the Watson–Crick face of the i-motif duplex self-associates into tetraplex via the formation of protonated C·C+ base pairs which requires acidic conditions. In the zipper-like motif, the central adenines do not form any hydrogen bonds with any other symmetry-related bases or phosphate groups, but rather make contacts mediated through a water molecule as mentioned above.

and was named the ‘base zipper’. There are, however, several striking differences between this RNA motif and the DNA motif reported here. As the base-zipper motif forms the core of the theophylline-binding pocket, there is an extensive network of hydrogen-bonding contacts to this motif; in particular, one base of the motif makes three hydrogen bonds to the theophylline and this residue is responsible for discriminating theophylline from caffeine. The phosphodiester chains of the base-zipper motif are disposed differently as well, with the two chains separated as far apart as possible. This disposition of the phosphodiester chains is more reminiscent of the intercalated packing mode in cyclic ribo-diguanylic and cyclic deoxyribodiadenylic acids [47,48].

The stacking of the adenine bases in the zipper-like motif differs somewhat from that seen in i-motifs (see Figure 3). In i-motifs, the hemiprotonated C·C+ base pairs crossstack in such a way that the intercalated base pairs are rotated almost 90° from each other, and which aligns only every other stack of pyrimidine rings. In the zipper-like motif, the stacking of intercalated purine rings and the bases of the flanking GCG sequences exhibits a small displacement of the base at each step, creating a sheared stack that winds its way around the duplex. A zipper-like motif has recently been discovered in an RNA structure that specifically binds theophylline [46]

Duplex or hairpin?

The X-ray crystallographic evidence presented here for a duplex structure of the oligonucleotide d(GCGAAAGCT) is not necessarily contradictory to the hairpin model proposed by Hirao et al. [10]. Rather, it is likely to be the duplex form of a dynamic equilibrium between hairpin and duplex conformations. Hairpin↔duplex transitions have been studied and shown to exist for a number of deoxyribose oligonucleotides, especially double-stranded

Research Article Structure of an adenine zipper in DNA Shepard et al.

DNA sequences containing inverted repeats or palindromes [49–54]. Non-self-complementary sequences are also known to undergo hairpin↔duplex transitions [21,24,55–57], and this includes the formation of pHdependent i-motifs [39,58–60]. Several parameters contribute to whether a given oligonucleotide folds into a hairpin structure or self-associates into a duplex. Most notably, these parameters include the nucleotide sequence, its length and its concentration (e.g. [24,57]), as well as salt concentration [55,56] and pH [39,44,58,59]. A detailed NMR study on variants of the d(NAATGNAATG) sequence (where N is either A, C, G or T), revealed that the oligonucleotides exist as differing equilibrium mixtures of single-residue hairpins and mismatched duplexes containing sheared G·A pairs and unpaired, intercalated bases [24]. Depending upon the central GNA sequence, for N = C, the strand folds exclusively into a hairpin. For N = A or T, the mixture contains approximately 20% duplex, and for N = G the mixture is predominately duplex. The preference of the N = G sequence to form duplexes, relative to the N = A sequence, which favours the hairpin form, is most probably a consequence of an additional guanine–phosphate hydrogen bond which contributes to the stability of the duplex form. Because adenine cannot form this particular hydrogen bond, it was reasoned that its duplex form is less stable. In the zipper-like motif, however, the corresponding adenine is in the same position as the guanine residue that interacts with the phosphate, and this suggests that the zipper-like motif is representative of the minor duplex form of d(NAATGNAATG) where N = A. In the NMR studies on the d(GCGAAGC) sequence [60] and its d(GCGNAGC) variants [61], which lack a central adenine compared with the sequence reported here, the NMR spectra did not demonstrate clear-cut duplex formation under experimental conditions. Reid and coworkers [23,24] suggested that the localised broadening in the spectra of the H1′ protons of the GAA loop of the d(GCGAAGC) sequence could just as well be due to a hairpin–duplex equilibrium as to the original interpretation of a small structural fluctuation caused by the wobbling of the single adenine loop residue. Although no duplex form was presented in the NMR studies on d(GCGAAAGC) [10], a hairpin–duplex equilibrium could very well exist for the d(GCGAAAGCT) sequence. As mentioned above, oligonucleotide and salt concentrations, along with the nucleotide sequence, are known to affect the relative hairpin–duplex populations of DNA oligomers. In general, the hairpin form is typically favoured at low oligomer and salt concentrations while the duplex form is favoured at high oligomer and salt concentrations (e.g. [55,56,62]). With this in mind, the appearance of hairpin or duplex structures of the d(GCGAAAGCT)

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fragment becomes more clear. The relatively high oligomer (and possibly salt) concentration in the crystallization solution probably favours the duplex form, and crystallization of the duplex form displaces the equilibrium as it is depleted from the mother liquor solution. In principle, crystallisation of the hairpin form is possible, but requires conditions where the hairpin is favoured over the duplex which must remain soluble during crystallization. Role of the cobalt hexammine cation

As stated earlier, the cobalt hexammine cation contributes more to crystal cohesion, as a result of bridging interactions between guanines of adjacent duplexes, than directly to duplex stability. The crystal stabilization effect of metal hexammine cations has been recognized in crystallizations of nucleic acid oligomers for some time [32,63–68], even though the cation itself may not be observed in the crystal structure [66]. In DNA, the cobalt hexammine cation has a well defined binding mode at the O6 or N7 sites on the major groove side of a guanine, often with an additional interaction to a phosphate oxygen [66,67,69,70]. In most cases, the cobalt hexammine sites cross-link between symmetry-related strands of the phosphate oxygens or guanine bases. Until recently [66], this cation has only been directly observed in association with DNA in its Z form, and in this particular case, the oligomer folds into an A DNA duplex. The only cobalt hexammine cation that was found not to bind to a guanine in a DNA crystal structure, instead binds to phosphate oxygens from adjacent duplexes [65]. In RNA structures, the cobalt hexammine is usually located in the major groove of the duplex, within a binding pocket built by tandem G·U base pairs [64,71], or linked in a similar way to a guanine step as observed in tRNA [72]. Similar binding modes are observed for other metal hexammines, such as osmium hexammine in RNA [64] or ruthenium hexammine in DNA [73]. The interest in cobalt hexammine-binding properties arises from the fact that it is known to promote the transition of either B to A or B to Z DNA conformations depending on the DNA sequence [63,67,68,74–77]. This cation is also effective in promoting the formation of the four-way (Holliday) junction [78]. In RNAs, the cobalt hexammine cation is known to mimic and substitute for hexahydrated magnesium cations, which are considered to be an important structural component in RNA molecules [64,71,72,79–81]. The use of these metal hexammine derivatives is thus a way of studying ribozyme catalytic behaviour [64]. The binding mode of cobalt hexammine in the structure presented here is similar to the most frequently observed binding mode where hydrogen-bonding interactions crosslink the O6 and N7 of a guanine step, except that three symmetry-related guanine bases are involved (Figure 4). Surprisingly, interactions with the surrounding phosphate

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groups are absent. The fact that the cobalt hexammine cation resides in a solvent channel and makes interduplex rather than intraduplex hydrogen bonds suggests that it does not affect the conformation of the zipper-like motif. Other structural studies on oligonucleotides also provide evidence for insignificant structural changes upon cobalt hexammine binding [66,71]. In light of the different structures analogous to the d(GCGAAAGCT) sequence, it would be interesting to find out if the zipper-like motif could extend beyond four intercalated and unpaired adenines, what factors would stabilise it over the hairpin form, and whether the motif is dependent upon the nucleotide sequence. At present, we are continuing research along these lines and, in particular, we have recently collected X-ray data to 1.7 Å resolution on isomorphous crystals containing iridium hexammine which will provide a more precise and detailed structure to be presented elsewhere. DNA secondary structure elements in replication origins and viruses

In the ssDNA bacteriophages α3, φK, G4 and St-1, the replication origins contain regions of significant nucleotide conservation that have been proposed, on the basis of sequence analysis, to fold into successive hairpin loops. These models are based primarily upon the formation of Watson–Crick base pairs, without considering other possible forms of DNA secondary structural elements. It is worth noting that in the vicinity of the replication origins of these bacteriophages, there exist several GA and GAA stretches which could potentially fold back on to each other and form intercalated or zipper-like motifs [3]. In particular, the replication origins of α3, φK and St-1 contain a tandem repeat with the GGAA consensus separated by only three residues. Tandem repeats of GRnA (where R is usually G or A) runs are also prevalent in the proposed replication origins of phages λ and φ80 [82,83], and of E. coli [84,85]. In phage λ, three GRnA tandem repeats exist ([GAAAA]2, [GAGGGA]2 and [GGGGGA]2) [82], in phage φ80, there are two GRnA tandem repeats ([GAAA]2 and [GAACA]2), and E. coli has only one GRnA tandem repeat ([GAATGA]2). Curiously, a GRnA tandem repeat occurs in or near the bubble in the stem of a proposed hairpin structure for these three cases [3]. Although ssDNA or dsDNA replication origins have the potential to fold into hairpin or cruciform structures, whether they do so in vivo still remains to be determined. The 3′ terminus of parvoviruses is a highly conserved sequence of 115 or 116 nucleotides which has been proposed to fold into a Y-shaped hairpin structure and which is also an origin for DNA replication [4,86]. There exists in the main stem of the Y-shaped hairpin a GAA:GA ‘bubble’ which is close to the initiation site of DNA replication and is resistant to mung bean endonuclease [4]. An unpaired

adenine base could be stacked between the guanine bases of two bracketing G·A mismatches, as in the zipper-like motif, and this could explain the endonuclease resistance of the GAA:GA bubble. Indeed, such a motif has been proposed on the basis of unpublished NMR studies by Chou et al. [27]. The zipper-like motifs and their transitions into hairpin structures might have an important role in viral replication. Recently, hairpin↔duplex transitions have been implicated in the initiation of DNA replication at palindromic telomeres of the minute virus of mice (MVM), a parvovirus [87]. Amplification of the replication form intermediate is initiated by the folding back of palindromic sequences which act as primers for strand-displacement synthesis. In fact, hairpin↔duplex transitions would aid parvovirus DNA replication where the folding and unfolding of palindromic sequences is necessary in the rolling hairpin model [86,88]. Furthermore, mismatched nucleotides of a bubble in the 5′-terminal hairpin of MVM were found to be critical for growth of the virus, but the nucleotide sequence was not critical [89]. The results suggest that the replacement of the bubble with full base pairs impairs the conversion of the hairpin 5′ termini to the extended form. This hints at the possibility of an unusual DNA secondary structure having a role in viral DNA replication. The zipper-like motif also provides a model for how filamentous inoviruses, which contain circular ssDNA, can package their genome. In the case of the inovirus Pf1, the interior cross-section diameter of the filamentous protein shell is too narrow to package standard B DNA [90–92]. To account for the genome packaging into the confined space of the interior of the Pf1 inovirus, models of a duplex with intercalated and unpaired bases were put forward on the basis of fibre diffraction studies, energy minimisation calculations and small-molecule crystallography [90,92,93]. In these models, however, it was reasoned that the negative charge on the phosphate groups would induce the two phosphodiester chains to separate as far apart as possible. The zipper-like motif shows that this restriction is not necessary, and that an extended zipperlike DNA conformation could act as a suitable model for the packaging of the genome of inoviruses. GAAA tandem repeats in centromeric DNA

Centromeric DNA of higher eukaryotes is characterised by large arrays of relatively short tandem repeats of untranscribed DNA which is associated with the kinetochore and is functionally important during mitosis and meiosis (for a review see [94]). Although the precise function of the tandem repeats in centromeres is obscure, it is thought that they are involved in the formation of a higher-order structural complex at the kinetochore [15,20,95]. A variety of centromeric DNA contains GAAA stretches or related

Research Article Structure of an adenine zipper in DNA Shepard et al.

sequences. (GGAAT)n repeats have been found to be highly conserved in the centromeres of humans and other higher eukaryotes [15]. The GAAA stretch is also found in the 17 bp CENP-B box which binds the CENP-B protein located in the centromere region beneath the kinetochore [18,96–99]. In the centromeres of fission yeast, which have been extensively studied as a model for higher eukaryote centromeres (for a review see [99]), the dg and dh repeat sequences and the central sequence are moderately rich in GAAA stretches [16,17,95]. Studies on centromere-like oligonucleotides have revealed that many of these sequences exhibit high thermal stability when separate from their complementary strands [15,21,100]. Unusual secondary structures, such as mismatched duplexes and fold-back structures containing G·A pairs and unpaired bases, have been implicated as being responsible for the exceptional stability of these ssDNA analogues in vitro [21–23,33]. In view of the nucleotide sequence similarities, the zipper-like motif presented here is probably one member of a family of unusual secondary structures in centromere-like DNA. Nevertheless, the existence for these unusual secondary structures in vivo, and whether they have a role in centromere function, depends upon the separation of the two complementary strands in dsDNA into ssDNA, which has, as yet, to be determined. But, it is interesting to note that the chromatin structure of the central core of centromeres from fission yeast has been reported to be unusual, with no evidence of regular nucleosomal packaging [94,95]. Furthermore, when the central core is spliced into a non-functional environment, the sequences are packaged normally into nucleosomes [94]. This has led to the suggestion that the unusual organisation of the core region is central to a higher-order structural complex that distinguishes the centromere from the chromatin arms. The basis for this might lie in unusual secondary structures of ssDNA which have been separated from their complementary strand by preferential binding with specialized nuclear proteins — a phenomenon observed with the centromeric dodeca-satellite DNA sequences from Drosophila [100]. Such unusual ssDNA secondary structures, controlled via the binding of the complementary strand, would open up the possibility of hairpins and mismatched duplexes containing zipper-like and [GGA]2 motifs with stacks of unpaired bases, and these structures would constitute a new family of DNA-binding sites available for interaction with proteins, such as those that make up the kinetochore complex.

Biological implications The fragment d(GCGAAAGCT) of the replication origin of bacteriophage G4 was previously proposed to form a hairpin [10]. In this paper, the fragment is shown instead to self-associate into a duplex containing a zipperlike motif —a novel secondary structure element in DNA. The intercalation and stacking of the central,

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unpaired adenines between two sheared G·A base pairs is a stable conformation that provides a way for noncomplementary strands of DNA to form a duplex. As such, it should be considered as a structural option when constructing the single-stranded (ss)DNA secondary structures of biological systems that contain tandem repeats of GAAA stretches or purine tracts. In particular, the replication origins of many bacteriophages contain tandem repeats of GRnA tracts often located in the stems of proposed hairpins [3]. Such repeats could provide an alternative to Watson–Crick fold-back structures in the form of a zipper-like duplex containing an array of stacked, unpaired purines. Furthermore, the exposed nature of the adenines in the zipper-like motif could allow proteins to bind to their free Watson–Crick face, and would constitute a new family of DNA-binding sites. The intercalated and stacked nature of the zipperlike motif also provides a model for the packaging of the circular ssDNA genome into the restricted space of the interior of filamentous inoviruses [90–93]. The polymorphism exhibited by many DNA sequences, such as the one studied here, is likely to be an important factor in the initiation of DNA replication in parvoviruses [86–88], where hairpin↔duplex transitions at the telomeres have been implicated in the rolling hairpin model. Indeed, Chou et al. [27] have proposed a zipperlike motif at the 3′ terminus of the parvovirus genome on the basis of unpublished NMR studies of the GAA:GA bubble, which is resistant to mung bean endonuclease. Similarly, the endonuclease resistance shown by other oligonucleotides could be explained by the formation of zipper-like motifs in mismatched duplexes. The GAAA consensus and similar analogues occur in a number of repetitive sequences of centromeric DNA [15–18,94–99]. Although the function of these tandem repeats in mitosis and meiosis is obscure there has been some speculation, on the basis of their exceptional thermal stability when separated from the complementary strand, that they have a structural role in the formation of unusual secondary structure. The existence of unusual secondary structure, such as hairpins or zipper-like duplexes, in vivo has yet to be determined, however.

Materials and methods Synthesis of the nonamer The nonanucleotide was synthesised by automated methods with an Applied Biosystem 391 synthesizer and phosphoroamidite monomers obtained from Millipore (a 5-Br cytosine was introduced at position 2). The product was hydrolysed with 3 M aqueous ammonia, washed with ether and precipitated with ethanol. Purifications were by reverse phase HPLC.

Crystallisation The nonamer dGCGAAAGCT (1 mg) was dissolved in 0.4 ml lithium cacodylate buffer (0.05 M). To this solution, 3 µl of 0.1 M NH4OH,

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15 µl of a 10 mM cobalt hexammine chloride solution and 8 µl of 50% methylpentanediol (MPD) solution were added. The solution was allowed to equilibrate at 4°C by vapour diffusion against a reservoir containing a solution of similar composition except with MPD at a higher concentration (25%). Small yellowish hexagonal crystals develop in one or two weeks. They are very stable at 4°C (diffracting well even after two years) but much less so at room temperature.

Data collection and processing Despite many attempts, it was not possible to solve the structure using molecular replacement methods with either a canonical B model or starting from different known B-DNA structures. Consequently, MAD data were collected about the bromine K-edge at four different wavelengths. Of the two crystals used, both were crystallographically aligned: one with the c* axis parallel and the other with the c* axis perpendicular to the spindle. The X-ray wavelengths were chosen from the X-ray fluorescence spectra of the bromine K-edge recorded directly from the crystals in order to optimise the anomalous dispersion effects from the bromine atom (Table 2). The MAD data were recorded with an 18 cm diameter image plate system on the new experimental station DW21b, situated at the end station of the LURE-DCI wiggler, Orsay, France. The crystal-detector distance was 210 mm and rotation frames of 2° were used for data collection. Exposure times per frame were 180–300 s. Diffraction images and data were processed using MOSFLM, SCALA and the CCP4 suite of programs [101]. Data were put on to a common scale in two steps: internal scaling was applied to the data of each wavelength of each crystal to correct for sample decay and incident beam fluctuations; and then data at all wavelengths were scaled as a smoothly varying function along the detector and spindle position utilising the long wavelength remote

(lowest f″) as a reference. Scale factors between the two crystals were determined at the heavy-atom refinement stage. Following the structure determination, a high-resolution data set was recorded on the large MAR Research image plate system (diameter 300 mm) available at the W32 wiggler beam line at LURE. The nominal resolution was 2.1 Å (for a ratio I/σ(I) = 4 in the last data shell 2.2–2.1 Å). This data set comprises 1832 independent structure factors over a total of 21,345 measured reflections (redundancy 11.6; Rsym 5.3%).

Phasing at the bromine edge Anomalous Patterson maps were calculated for each individual wavelength and crystal [102,103], as well as for the set of |oFA| as determined from algebraic equations implemented in the program MADLSQ [104]. This allowed us to follow the evolution of the Patterson peaks in the maps with respect to the change in wavelength. Only one distinct set of peaks appears in the Patterson maps. The corresponding site and its enantiomorph were entered as the bromine atom of the cytosine for refinement using the program SHARP [105] and then followed by solvent flattening with the program SOLOMON [106]. Choice of the enantiomorph was made by inspecting the electron-density maps and the correct one was clearly evident showing the macromolecular boundary and the stacking of bases along the c axis. Eight out of nine of the bases were easily identified and could be fitted into density, but tracing of the phosphate backbone was unclear at certain points. Along the crystallographic threefold axis, however, a cobalt hexammine was readily identified as it is known to be necessary for the growth of the crystals. After including this cobalt and its anomalous scattering as an additional ‘fragment’ into the second round of heavy-atom refinement

Table 2 Crystal data. Crystal 1 (c* perpendicular to spindle) Wavelength (Å)

Resolution limits (Å) Nobs Nuniq Redundancy Completeness (%) I > 3σI (%) Rsym* Ranom† RCullis (centrics)‡ Dispersive phasing power (centrics/acentrics)§ Anomalous phasing power (acentrics) Theoretical f′/f″ ¶ Experimentally refined f′/f″ #

Crystal 2 (c* parallel to spindle)

0.9119 short λ remote

0.9191 peak

0.9200 edge

0.9536 long λ remote

0.9119 short λ remote

0.9191 peak

0.9196 edge

0.9535 long λ remote

33.4–2.43 15,433 1299 11.9 100 90.2 0.045 0.052 0.893

33.4–2.43 16,218 1297 12.5 100 93.1 0.037 0.056 0.611

33.4–2.43 15,153 1297 11.7 100 89.6 0.048 0.029 0.736

33.4–2.45 13,653 1261 10.8 97.2 92.2 0.034 0.020 0.710

28.9–2.33 14,526 1287 11.3 96.6 91.0 0.052 0.051 0.744

28.9–2.36 11,182 1231 9.1 92.2 88.7 0.054 0.056 0.663

28.9–2.36 10,929 1208 9.0 90.3 88.1 0.052 0.034 0.646

28.9–2.46 6357 1076 5.9 79.8 87.4 0.048 0.025 0.707

2.35/3.65

1.92/3.25

1.11/3.02

1.15/2.90

1.47/2.49

1.89/3.34

2.02/3.11

1.47/2.91

4.84 –4.122/ 3.752 –4.122/ 3.752

4.30 –6.380/ 3.814 –10.931/ 8.186

2.74 –9.634/ 2.163 –14.687/ 2.913

1.93 –2.811/ 0.543 –2.811/ 0.543

2.52 –4.122/ 3.752 –4.122/ 3.752

4.83 –6.380/ 3.814 –13.604/ 4.561

2.66 –9.634/ 2.163 –9.486/ 1.952

1.85 –2.977/ 0.536 –2.977/ 0.536

The crystal is in space group P6322 with unit-cell dimensions a = b = 37.56 Å and c = 65.39 Å. There is one strand per asymmetric unit corresponding to a volume of 739 Å3/residue; the calculated solvent content is approximately 36%. *Rsym = ΣhklΣi |Ii – 〈I+/–〉| / Σhkl 〈I+/–〉. †Ranom = Σhkl |〈I+〉 – 〈I–〉| / Σhkl (〈I+〉 + 〈I–〉) for I+ and I– on the same or adjacent images. ‡RCullis = 〈phase-integrated lack of closure〉 / 〈|FPH – FP|〉. §Phasing power = 〈{|FH(calc)|/ phase-integrated lack of

closure}〉. ¶Theoretical f′ and f″ values estimated from [109]. #Experimentally refined values of f′ and f″ were calculated during the heavy-atom refinement procedure in SHARP. The f′ and f″ values were held constant for wavelengths remote from the Br K-edge, and they are strongly correlated with other parameters in the heavy-atom refinement (e.g. occupancy, thermal factors, scaling between crystals, resolution, etc).

Research Article Structure of an adenine zipper in DNA Shepard et al.

with SHARP, the tracing of the entire DNA molecule including the phosphate backbone became trivial. The exceptional quality of the map is such that the zipper-like motif became unambiguous and even some structural water molecules could be clearly seen. The initial model was built using an interactive graphics interface.

Refinement of the structure The refinement was performed with the data set recorded at high resolution (2.1 Å). Beyond the last phosphate group (position 9) the electron density is too feeble to build the last base, a thymine, which is disordered as it points towards a threefold axis. A first round of refinement was conducted with XPLOR [107] at the maximum resolution then with the SHELX97 program [108]. Water molecules were introduced during the course of the refinement following electron-density map inspections on a graphics system. The final model comprises 185 atoms (including the disordered thymine), a third of a cobalt hexammine cation, a chlorine ion and 18 water molecules. The R factor is 18.9% for all data (1832 structure factors); the corresponding Rfree is 24.1%. The root mean square (rms) bond distances and angles are 0.015 Å and 2.4°, respectively.

Accession numbers The coordinates of the d(GCGAAAGCT) structure have been deposited with the Nucleic Acid Data Bank (accession code UDIB70) and are on hold for one year.

Acknowledgements We thank F Fossard, C Arrachart and C Maman for assistance during data collection and processing. We are also especially grateful to D Ragonnet and D Chandesris for aid and support on the DW21 beam line. We are grateful to Olga Kennard and the members of the CCDC laboratory (Cambridge, UK) where WBTC initiated this project.

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