An A-type double helix of DNA having B-type puckering of the deoxyribose rings1

doi:10.1006/jmbi.2000.3592 available online at http://www.idealibrary.com on

J. Mol. Biol. (2000) 297, 907±922

An A-type Double Helix of DNA Having B-type Puckering of the Deoxyribose Rings LukaÂsÏ TrantõÂrek2,3, Richard SÏtefl2,4, Michaela VorlõÂcÏkovaÂ1 Jaroslav KocÏa2,3, VladimõÂr SklenaÂrÏ2,4 and Jaroslav Kypr1* 1

Institute of Biophysics of the Academy of Sciences of the Czech Republic, KraÂlovopolska 135, CZ-612 65 Brno, Czech Republic 2

Laboratory of Biomolecular Structure and Dynamics Masaryk University KotlaÂrÏska 2, CZ-611 37 Brno, Czech Republic 3

Department of Organic Chemistry, Masaryk University, KotlaÂrÏska 2, CZ611 37 Brno, Czech Republic 4

Department of Theoretical and Physical Chemistry, Masaryk University, KotlaÂrÏska 2, CZ611 37 Brno, Czech Republic

DNA usually adopts structure B in aqueous solution, while structure A is preferred in mixtures of tri¯uoroethanol (TFE) with water. However, the octamer d(CCCCGGGG) and other d(CnGn) fragments of DNA provide CD spectra that suggest that the base-pairs are stacked in an A-like fashion even in aqueous solution. Yet, d(CCCCGGGG) undergoes a cooperative TFE-induced transition into structure A, indicating that an important part of the aqueous duplex retains structure B. NMR spectroscopy shows that puckering of the deoxyribose rings is of the B-type. Hence, combination of the information provided by CD spectroscopy and NMR spectroscopy suggests an unprecedented double helix of DNA in which A-like base stacking is combined with B-type puckering of the deoxyribose rings. In order to determine whether this combination is possible, we used molecular dynamics to simulate the duplex of d(CCCCGGGG). Remarkably, the simulations, completely unrestrained by the experimental data, provided a very stable double helix of DNA, exhibiting just the intermediate B/A features described above. The double helix contained well-stacked guanine bases but almost unstacked cytosine bases. This generated a hole in the double helix center, which is a property characteristic for A-DNA, but absent from B-DNA. The minor groove was narrow at the double helix ends but wide at the central CG step where the Watson-Crick base-pairs were buckled in opposite directions. The base-pairs stacked tightly at the ends but stacking was loose in the duplex center. The present double helix, in which A-like base stacking is combined with B-type sugar puckering, is relevant to replication and transcription because both of these phenomena involve a local B-to-A transition. # 2000 Academic Press

*Corresponding author

Keywords: DNA; A-type double helix; B-type deoxyribose pucker; buckled CG step; minor groove widening

Introduction It has been shown by Franklin & Gosling (1953) that DNA can adopt two ordered structures, designated A and B, and switch between them depending on humidity. In solution, the B-A transition has subsequently been induced by ethanol (Brahms & Mommaerts, 1964; Ivanov et al., 1974; Abbreviations used: TFE, tri¯uoroethanol; NOESY, nuclear Overhauser spectroscopy; TOCSY, total correlated spectroscopy; DQF-COSY, double quantum ®ltered correlated spectroscopy; HSQC, heteronuclear single-quantum correlation; MD, molecular dynamics; RMSD, root-mean-square deviation; PME, particle mesh Ewald. E-mail address of the corresponding author: mi®@ibp.cz 0022-2836/00/040907±16 $35.00/0

Zimmermann & Pheiffer, 1979; Kypr et al., 1986), methanol (VorlõÂcÏkova et al., 1984), tri¯uoroethanol (Ivanov & Minyat, 1981; VorlõÂcÏkovaÂ, 1995) and multivalent cations (Minyat et al., 1978; Xu et al., 1993; Robinson & Wang, 1996). The A structure is biologically relevant because it is an almost constitutive conformation of double-stranded RNA and it is induced in DNA by polymerases (Florentiev & Ivanov, 1970; Beabealashvily et al., 1971; Eom et al., 1996) and other proteins (reviewed by Setlow, 1992; Ivanov et al., 1995; Jones et al., 1999). The stability of A-DNA is most promoted by the d(CC)
d(GG) steps (Minchenkova et al., 1986; Mazur et al., 1989; Foloppe & Mackerell, 1999) while the runs of A in one strand bound to runs of T in the complementary strand are reluctant to isomerize into A-DNA (Pilet et al., 1975; Becker & # 2000 Academic Press

908 Wang, 1989). The B-A transition is cooperative, which means that double helices intermediate between structure B and structure A are not stable. The reasons for the instability of the intermediate B/A double helices are unknown (Calladine & Drew, 1984; Heinemann et al., 1990; Tung, 1992; Marky & Olson, 1994; Cheatham et al., 1997). This work deals mainly with the DNA duplex of d(CCCCGGGG). This octamer, as well as other members of the d(CnGn) family of oligonucleotides, produce DNA duplexes even in aqueous solution whose CD spectra contain the strong band in the vicinity of 260 nm that is a typical feature of structure A (VorlõÂcÏkova et al., 1984). The d(CCGG) (Matsuzaki et al., 1986), d(CCCGGG) (Wolk et al., 1989) and d(CCCCGGGG) (Benevides et al., 1986) duplexes have been studied by various methods. The features provided by these studies and con®rmed here include Watson-Crick pairing of the bases and B-type sugar puckering (Benevides et al., 1986; Wolk et al., 1989). The d(CCCCGGGG) duplex is composed of two important elements. First, it is the d(CCCC)
d(GGGG) duplex constituting both octamer duplex halves. The d(C)n
d(G)n duplex of DNA is known to be unwound (Biburger et al., 1994) and prone to switch into structure A easily (Arnott & Selsing, 1974; Nishimura et al., 1986; Sarma et al., 1986; Warne & deHaseth, 1993). The other important element of the duplex of d(CCCCGGGG) is the central CG step. This step is interesting from the biological (reviewed by Colot & Rossignol, 1999) and the structural point of view (Haran et al., 1987; Heinemann et al., 1987; Mauffret et al., 1989; Takusagawa, 1990; SÏponer & Kypr, 1990, 1993; El antri et al., 1993; Lefebvre et al., 1995a,b; Chaoui et al., 1999; Cordier et al., 1999). We have been studying DNA conformation by CD spectroscopy for many years and hence know that CD spectroscopy is a very sensitive and reliable indicator of base stacking in DNA. That is why we have been concerned with the question for a long time of how the B-type sugar pucker can be combined with A-like base stacking in the DNA duplexes of d(CCCCGGGG) and similar d(CnGn) oligonucleotides. The work described here provides an answer to this question.

Results CD spectroscopy Figure 1 shows CD spectra of the DNA octamer d(CCCCGGGG) and tetramer d(CCGG) in a lowsalt aqueous buffer. These spectra are characterised by the strong positive band in the vicinity of 260 nm and by the negative band in the vicinity of 280 nm. Qualitatively the same spectra are generally provided by DNA duplexes of the d(CnGn) fragments (M. V., unpublished results). The qualitative difference from the canonical B-form is demonstrated here with d(CGCGCGCG), a repre-

An Intermediate B/A Duplex of DNA

Figure 1. CD spectra of (bold trace) the duplex of d(CCCCGGGG), (- * -) denatured single strand of d(CCCCGGGG) at 84  C, ( Ð ) duplex of d(CCGG), and (- -) duplex of d(CGCGCGCG). The duplexes were all measured at 0  C. All three oligonucleotides were dissolved in 1 mM sodium phosphate, 0.3 mM EDTA, pH 7. Inset: Thermal melting of the duplex of d(CCCCGGGG) monitored through the elliplicity changes at 262.5 nm.

sentative of the canonical B-DNA. The unusual CD spectrum of d(CCCCGGGG) has a conformational origin, because the CD spectrum is qualitatively different upon thermal denaturation and simultaneously the same as with various other DNAs. Thermal melting of the duplex of d(CCCCGGGG) is surprisingly cooperative for such a short molecule (Figure 1). Figure 2 shows the tri¯uoroethanol (TFE)induced transition of d(CCCCGGGG). The resulting CD spectrum is typical for the A-form with a strong CD band at around 260 nm and a negative band in the vicinity of 210 nm (VorlõÂcÏkovaÂ, 1995). The transition is quite cooperative, so that the aqueous duplex of d(CCCCGGGG) is evidently different from the A-form that is stable at about 75 % (v/v) TFE. On the other hand, the high degree of similarity between the aqueous and TFE spectra is unusual because the aqueous conformers of DNA mostly look like that of d(CGCGCGCG) in Figure 1. This similarity suggests that the aqueous and TFE conformers of d(CCCCGGGG) share the A-like geometry of base stacking. This raises the question of how the A-like stacks of base-pairs are accommodated into the aqueous double helix of d(CCCCGGGG). This question is answered here by a combination of NMR spectroscopy and molecular dynamics.

909

An Intermediate B/A Duplex of DNA

Figure 2. CD spectra of the duplex of d(CCCCGGGG) during its TFE-induced transition into structure A. TFE was added to the sample of d(CCCCGGGG) that was originally dissolved in 10 mM sodium phosphate, 0.3 mM EDTA, pH 7. Temperature, 0  C. The CD spectra were measured in the presence of (- - -) 60 % TFE, ( Ð ) 70 % TFE, and ( Ð ) 80 % TFE. Inset: The TFEinduced transition in d(CCCCGGGG) monitored through the ellipticity changes at 270 nm.

NMR spectroscopy The sequential NOE connectivities of nonexchangeable protons of the duplex of d(CCCCGGGG), their relative intensities (data not shown) as well as the backbone torsion angles are consistent with a right-handed DNA double helix (WuÈthrich, 1986). A continuous train of connectivities was detected, linking the imino and amino protons of non-terminal base-pairs. The connectivities of the imino-to-amino protons, as well as the chemical shifts of the imino protons, indicate Watson-Crick base-pairing. In addition, the carbon and phosphorus chemical shifts display the normal dispersion found in regular right-handed helical structures (Gorenstein, 1994; Wijmenga & Buuren, 1998). The values of sugar pucker parameters extracted from NMR spectra (for details, see Materials and Methods) are summarized in Table 1. The parameters show that the S-type sugar conformation typical for B-DNA, but absent from A-DNA, is dominant in all residues. Signi®cant deviations were found only at the central C4 and G5, which both exhibited substantially smaller ranges of the pseudorotation phase angles PS (C4, 123.5-144.1; G5, 98.5-100.4).

The exocyclic torsion angles (a, b, g, z, e, w) of the phosphodiester backbone were determined as described in Materials and Methods. The torsion angle restraints extracted from NMR spectra are presented in Figure 3 along with the results of molecular dynamics (MD) simulations (see below). The values of torsion angles w and b fall into the anti and trans conformational regions for all residues. In the course of the a, g, z, and e torsion angle analysis, the a $ g crankshaft motion and the in¯uence of BI/BII equilibrium on e and z was considered. The BI/BII equilibrium was assessed from the proton-phosphorus three-bond coupling constants 3J(H30 -P) and the 31P chemical shifts (Figure 4). Although the 31P chemical shift dispersion did not exceed the range expected for regular DNA structures, signi®cant down®eld 31P chemical shifts were observed for the C4 and G5 phosphorus atoms. The down®eld shifts, together with the substantially higher 3J(H30 -P) scalar coupling constants (8.3 and 8.8 Hz), indicate a contribution from the BII state (Gorenstein, 1994). This is a reason why the ranges for the e and z torsion angles include both trans/ÿ sc and ÿsc/trans regions at the central CG step of the d(CCCCGGGG) duplex. More complicated situations have been encountered in analyzing the a $ g crankshaft motions (correlated transitions of a and g from the (gaucheÿ, gauche‡) to (trans, trans) conformations). In principle, the restraint for the a torsion angle can be monitored only indirectly through the 31P chemical shift. However, the stereoelectronic effect of the a torsion angle on the 31P chemical shift cannot be separated from the stereoelectronic contribution of the z torsion angle in the presence of BI/BII equilibrium. As a result of severe signal overlap, the values of the g torsion angles were estimated only from H40 -H50 and H40 -H500 passive scalar couplings observed in the 2D 1H-31P correlation spectra. Since the presence of a $ g crankshaft motion cannot be detected unambiguously by NMR spectroscopy, wider ranges for the a and g torsion angles are presented in Figure 3 and considered in the analysis. A striking tendency was observed in the sugarto-base inter-residual distances by a quantitative analysis of the NOE data (Table 2). The intrastrand H6/8(n ‡ 1)-to-H10 (n) distances became Ê ) from the ®rst to consecutively longer (3.2-3.8 A the fourth cytosine base (C1-C4). This distance was Ê even in the central CG step. The distances 4.0 A showed an opposite trend from G5 to G8, where Ê ). they consecutively became shorter (3.5-3.3 A Molecular dynamics The d(CCCCGGGG) duplex was subjected to three unrestrained MD simulations, with the total length of over 16 nanoseconds. The ®rst simulation started from the canonical A-DNA duplex (Arnott & Hukins, 1972), the second from the canonical B-DNA duplex (Arnott & Hukins, 1973) and the third from the X-ray crystal structure of

910

An Intermediate B/A Duplex of DNA

Table 1. Pseudorotation phase angles (PS, PN), the pucker amplitudes (fS, fN) and fraction fS of the S-type conformer of the deoxyribose rings in the d(CCCCGGGG) duplex at 20  C Residue C1 C2 C3 C4 G5 G6 G7 G8

PS

PN

fS

fN

fS (%)

123.0-153.8 155.4-157.0 173.0-174.4 123.5-144.1 98.5-100.4 122.0-165.0 148.0-151.7 177.0-186.0

26 26 26 26 19 19 19 19

36 36 36 36 36-40 33-36 36-39 34-36

36 36 36 36 36 36 36 36

88.1-89.0 87.0-87.5 96.0-97.1 86.0-88.9 91.0-93.8 93.1-100 87.0-89.9 81.0-84.6

Pseudorotation parameters were determined from the sugar ring interproton three-bond scalar coupling constants by program PSEUROT.

d(CCCCGGGG) (NDB code ADH012; Haran et al., 1987). The lengths of the simulations were 6.8, 7.1, and 2.6 ns, respectively, but all of the simulations have already converged within about 0.4 ns to stable and similar conformational ensembles (Figure 5). After the convergence of trajectories, the structure remained relatively conserved during the rest of the simulations. However, the stable trajectories displayed signi®cant motions and departures from the average structure. These deviations mainly resulted from the BI to BII transitions and crankshaft motions of the central CG step, as discussed below. Of particular interest was the central hole in the resulting double helix, which is a feature absent from B-DNA but characteristic for A-

DNA (Conner et al., 1984; Heinemann et al., 1990). The cytosine bases were unstacked (slide about Ê ) in the simulated double helix, while the ÿ2.1 A six-membered ring of the 30 -end guanine base was positioned over the ®ve-membered ring of the 50 Ê with respect end guanine base (slide about ÿ1.9 A to the local helical axis). The ®ve-membered ring of guanine stacked over cytosine in the central CG step. In addition, interstrand stacking of the sixmembered rings of guanine was observed as a transient conformation of the CG step (vide infra). The RMSD values between various relevant structures are summarized in Table 3. The smallest RMSD between the structure, averaged over the last nanosecond of the individual simulation and

Figure 3. Exocyclic torsion angle restraints derived from MD and NMR.

911

An Intermediate B/A Duplex of DNA

Figure 4. Dependence of the 31P chemical shifts (&) and the 3JH30 -P coupling dispersion (*) on the nucleotide position in the d(CCCCGGGG) duplex at 20  C (pH 7.4, 1.95 mM strand concentration).

the starting conformation, was found for the canonical A-DNA. The RMSD values suggested that the overall conformation of the simulated double helix was A-like, as discussed below. The average conformational parameters of the d(CCCCGGGG) duplex are presented in Table 4. The simulated structures of d(CCCCGGGG) show stable stacking of the terminal bases. The structures differ mainly at the central CG step. The BI-BII transitions and the a . g crankshaft motions were found to be responsible for this ¯exibility. The central CG step is characterized by the enhanced ¯exibility and thermal mobility with respect to the other residues. It shows large deformability and deviations from the canonical geometry. In the course of all simulations, the BIBII transitions were observed. The torsion angles e and x of mainly the G, but also C, dodged synchronically from (trans, gaucheÿ) to (gaucheÿ, trans). The BII conformation was observed for about 35 % of the simulation time. The BI-BII transitions were sometimes coupled to the a . g crankshaft motions. The central CG step showed a low rise, low helical twist and larger roll when compared to the

other parts of the molecule. As mentioned above, the interstrand guanine stacking was a transient state of the CG step (Figure 6). Simultaneously, the cytosine bases almost completely lost the overlap with the guanine bases. The interstrand G-G stacking resulted from the positive shift and negative slide of the guanine bases. The stacking geometry observed here is very similar to that seen in the ADNA crystal structures of d(CCCCGGGG) (Haran et al., 1987) and d(GGGCGCCC) (Shakked et al., 1989) as well as in various oligonucleotide duplexes in aqueous solution (Mauffret et al., 1989; El antri et al., 1993; Lefebvre et al., 1995a,b, 1996). The interstrand guanine stacking was characteristic for one geometry of the central CG step observed during our MD simulations of d(CCCCGGGG). The second characteristic geometry had a transient BII conformation of the backbone and an extremely wide minor groove. The value of the x-displacement from the double helix center was relatively Ê ) with all conserved in the CG step (about ÿ3.9 A simulated double helices of d(CCCCGGGG) (Figure 7). This is almost exactly the same value as the average value of this parameter observed in the A-DNA crystal structures (Young et al., 1997).

Table 2. H10 (n) to H6/8(n ‡ 1) intrastrand interproton distances in the d(CCCCGGGG) duplex in its canonical structure A, canonical structure B, the crystal structure of d(CCCCGGGG), the MD simulation of d(CCCCGGGG) and the NMR of d(CCCCGGGG) Ê) di (A a

Canonical A Canonical Bb X-rayc MD NMRd a

C1-C2

C2-C3

C3-C4

C4-G5

G5-G6

G6-G7

G7-G8

4.05 2.80 4.47 4.10 3.23

4.05 2.80 4.68 3.75 3.68

4.05 2.80 4.43 3.83 3.80

4.11 2.82 4.87 4.55 4.03

4.11 2.82 4.61 3.85 3.51

4.11 2.82 5.06 3.79 3.17

4.11 2.82 4.99 4.06 3.34

Arnott & Hukins (1972). Arnott & Hukins (1973). c Haran et al. (1987). d Distances were derived from a 2D NOESY experiment in 2H2O (the mixing time of 150 ms). Distance errors are approximately Ê for 2.0-3.5 A Ê , 0.4 A Ê for 3.5-4.5 A Ê and 0.5 A Ê for distances >4.5 A Ê. 0.3 A b

912

An Intermediate B/A Duplex of DNA

Figure 5. A stereoview of the superimposed average structures over the last nanosecond of the MD simulation of d(CCCCGGGG) starting from the canonical structure A (Arnott & Hukins, 1972) and from the crystal structure of d(CCCCGGGG) (Haran et al., 1987). The RMSD between these Ê. two structures is only 0.39 A

B-DNA has this parameter close to zero. The nonzero x-displacement re¯ects the central hole seen along the long helical axis of the double helix (Figure 5(b)). Another parameter that is close to zero in BDNA but non-zero in A-DNA is the base-pair inclination. In the present duplex of d(CCCCGGGG), the inclinations are larger in the central CG step than in the rest of the molecule. The most signi®cant property of the central CG step probably is buckling of the constituent base-pairs. The buckles are large and opposite in sign (Figure 7). Propeller twists are slightly positive at the central CG step, while the propellers are mostly negative in the crystal structures of DNA (Drew et al., 1981). The

double helix loosens towards the central CG step, whose deformation is accompanied by underwinding. The values of helical parameters show that the different conformations of the backbone at the CG step do not necessarily induce substantial variations in the helical parameters. The hydration of the major and minor grooves (data not shown) of the d(CCCCGGGG) duplex are very weak in comparison to nucleotide sequences containing adenine and thymine. In addition, no sodium ions were found in the major or minor grooves of the present d(CCCCGGGG) duplex. The unrestrained MD simulations of the d(CCCCGGGG) indicate that the minor groove is progressively widened towards the central CG step (Table 5).

Ê ) of the heavy atoms between the indicated structures Table 3. RMSD (A

Crystal structurea Canonical ADNAb Canonical B-DNAc X-ray startd A-DNA starte a

Canonical A-DNAb

Canonical B-DNAc

Crystal startd

A-DNA starte

B-DNA startf

3.48

4.37

3.44

3.47

3.62

4.31

2.50 3.00

2.44 3.17 0.39

2.88 2.83 0.94 0.90

of d(CCCCGGGG) Haran et al. (1987). of d(CCCCGGGG) Arnott & Hukins (1972). c of d(CCCCGGGG) Arnott & Hukins (1973). d Average structure over the last nanosecond of the MD trajectory starting from the X-ray crystal structure of d(CCCCGGGG) (Haran et al., 1987). e Average structure over the last nanosecond of the MD trajectory starting from the canonical A-DNA of d(CCCCGGGG) (Arnott & Hukins, 1972). f Average structure over the last nanosecond of the MD trajectory starting from the canonical B-DNA of d(CCCCGGGG) (Arnott & Hukins, 1973). b

913

An Intermediate B/A Duplex of DNA

Figure 6. A stereoview snapshot of the central CpG step from the MD simulation of the d(CCCCGGGG) duplex. The Figure shows the interstrand guanine stacking.

Comparison of the NMR and MD data The sequential resonance assignments of the 2D H NOESY spectra recorded in H2O and 2H2O at different mixing times, as well as the extracted inter-residual inter-proton distances indicated that d(CCCCGGGG) was associated into a righthanded, double-helical structure containing the conventional Watson-Crick base-pairs. The Watson-Crick base-pairs were stable in the course of the MD simulations, without any base-pair fraying at the double helix termini. NMR data (the corresponding intra-residue inter-proton distances and the appropriate three-bond scalar coupling constants) suggests that the S-type sugar geometry is dominant for all residues in the d(CCCCGGGG) duplex. The same results were obtained from the MD simulations. The central CCGG section of the octanucleotide shows deviations of the exocyclic torsion angles a, g, e and z in both the NMR and MD data. The lower helical twist seen in the MD simulations is probably re¯ected in the chemical shift deviations of the corresponding 31P NMR resonances and in the variations of the appropriate 3JH30 -P coupling constants (Figure 4). In general, the torsion angle ranges sampled during the MD simulations ®t the NMR-derived restraints very well (Figure 3). The BI-BII transitions at the CG step were indirectly detected in the NMR spectra using the H30 -P scalar coupling constants and the 31P chemical shifts, as discussed above. A semi-quantitative analysis of 1

the H30 -P coupling constants (Gorenstein, 1994) indicates nearly equal populations of the BI and BII states at the central CG step. This is consistent with the MD simulations, where population of the BII conformation was 35 %. The NOE spectra allow for measurements of distances between the 50 -end sugar H10 and the neighboring 30 -end base (H8 or H6) protons. These distances are in qualitative agreement with the MD simulations and closer to the value characteristic for the canonical A-DNA rather than B-DNA (Table 2). The decisive helical parameters, i.e. XDP, RIS and TWS (Figure 7), also suggest that the d(CCCCGGGG) double helix belongs to the family of A-DNA rather than B-DNA. Another remarkable feature of the present double helix of d(CCCCGGGG) that was identi®ed by the unrestrained MD, is the opposite buckle of the base-pairs in the central CG step (Figure 7). This buckle is re¯ected also in the H10 (n)-to-H6/8 (n ‡ 1) distances, which both the NMR and MD data suggest to be longer at the CG step than in the rest of the molecule. In the averaged MD structures, these distances are slightly longer in comparison with the values resulting from the NMR experiments. This could be due to the Cornell et al. (1995) force-®eld, which underestimates the C20 -endo sugar pucker and helical twist (Cheatham et al., 1997). As a result of the preference for the C10 -exo conformation, the H10 (n)-to-H6/8 (n ‡ 1) distances are probably overestimated in the MD data.

Table 4. The average backbone torsion angles, glycosidic angles, pseudorotation phase angles, and amplitudes calculated for different geometries of the d(CCCCGGGG) duplex Alpha (deg.) Beta (deg.) Gamma (deg.) Delta (deg.) Epsilon (deg.) Zeta (deg.) Chi (deg.) Phase (deg.) Amplitude

Canonical B

Canonical A

B-DNA starta

A-DNA startb

Crystal startc

Crystald

313.2 (0.0) 214.0 (0.0) 36.4 (0.0) 156.4 (0.0) 155.0 (0.0) 264.9 (0.0) 262.1 (0.1) 191.6 (0.1) 36.3 (0.0)

276.1 (0.0) 207.9 (0.0) 45.5 (0.0) 84.3 (0.0) 179.5 (0.0) 311.0 (0.0) 205.8 (0.1) 13.1 (0.0) 39.0 (0.0)

276.7 (48.2) 174.4 (8.3) 64.8 (36.3) 114.6 (5.1) 194.0 (38.9) 264.6 (41.5) 227.4 (8.8) 120.3 (8.4) 35.2 (3.0)

277.3 (5.5) 174.7 (8.3) 64.6 (6.7) 115.1 (5.7) 193.2 (7.2) 266.1 (6.1) 227.3 (8.5) 122.0 (9.1) 36.6 (2.1)

277.8 (6.4) 174. (7.8) 64.6 (10.1) 114.9 (6.5) 193.5 (8.0) 266.2 (5.5) 227.1 (8.1) 122.2 (8.9) 36.7 (2.0)

266.2 (52.3) 177.6 (13.8) 83.7 (47.1) 89.4 (17.3) 204.5 (13.1) 288.9 (10.0) 198.3 (13.2) 83.3 (115.8) 37.3 (3.7)

For details, see the text. The backbone torsion angles: O30 -P-a-O50 -b-C 50 -g-C40 -d-C30 -e-O30 -z-P-O50 (Saenger, 1984). The standard deviations are displayed in parentheses. a Average structure over the last nanosecond of the MD trajectory starting from the canonical B-DNA (Arnott & Hukins, 1973). b Average structure over the last nanosecond of the MD trajectory starting from the canonical A-DNA (Arnott & Hukins, 1972). c Average structure over the last nanosecond of the MD trajectory starting from the X-ray crystal structure (Haran et al., 1987). d Crystal structure of d(CCCCGGGG) (Haran et al., 1987). The values are taken from the NDB database (NDB code ADH012).

914

An Intermediate B/A Duplex of DNA

Discussion

Figure 7. Helical parameters of the average structures calculated with the Dials and Windows (Ravishanker et al., 1989) interface to Curves (Lavery & Sklenar, 1988). The average structures of the d(CCCCGGGG) duplex over the last nanosecond of the simulation starting from the canonical A-DNA (Arnott & Hukins, 1972) (bold line) and from the canonical B-DNA (Arnott & Hukins, 1973) (broken line). The x-displacement (XDP) is given Ê , inclination (INC) in degrees, buckle (BKL) in in A degrees, propeller twist (PRP) in degrees, opening Ê , tilt (TLT) in degrees, (OPN) in degrees, rise (RIS) in A roll (ROL) in degrees, and twist (TWS) in degrees.

The genetic information of all known free organisms is deposited in molecules of double-stranded DNA that mostly adopt structure B under physiological conditions. However, DNA can switch into structure A, which is an almost constitutive conformation of RNA. RNA probably preceded DNA in evolution (Jeffares et al., 1998) so that the basic mechanisms of genetic information copying are likely to have evolved on structure A rather than structure B. This view is consistent with the fact that many polymerases switch the template DNA into structure A locally at positions of genetic information copying (Wlassoff et al., 1996; Bebenek et al., 1997; Doublie et al., 1998; Kiefer et al., 1998). Thus, DNA switching into structure A may in¯uence replication and transcription of the genomes of the current organisms, which could have far-reaching consequences. In addition, numerous reports have been published concerning intermediate B/A structures of DNA of various kinds (Borden, 1993; Pavletich & Pabo, 1993; Cho et al., 1993; CruzeiroHansson et al., 1994; Ding et al., 1996, 1998; Bachelin et al., 1997; Tonelli et al., 1998; Malinina et al., 1999). The B-A transition depends on the nucleotide sequence, which may mean that some sequences are copied better (or worse) than others. The strongest manifestation of this dependence is exhibited by runs of adenine bases (thymine in the complementary strand) that are totally reluctant to switch into structure A (Pilet et al., 1975; Becker & Wang, 1989), while runs of cytosine bases (guanine in the complementary strand) switch into structure A very easily (Minchenkova et al., 1986). This ease of switching into structure A likely is caused by the unusual structure lying midway between ADNA and B-DNA, as shown here and in previous unrestrained MD simulation of (dG)10
(dC)10 (Cheatham et al., 1998). Experimental studies have demonstrated that in solution stretches of oligo(dG)
oligo(dC) unwind DNA to the A-like value of 11.1 base-pairs per turn of the double helix (Biburger et al., 1994). Poly(dG)
poly(dC) switches into structure A even in the absence of alcohol (Arnott & Selsing, 1974; Nishimura et al., 1986; Sarma et al., 1986) needed to stabilize other sequences in structure A. Guanine-rich sequences are recognized in DNA by the transcription factor IIIA through their RNA-like conformation (Rhodes & Klug, 1986). This aspect was a matter of controversy in the literature (McCall et al., 1986; Gottesfeld et al., 1987; Fairall et al., 1989; Galat, 1990; Huber et al., 1991) because the guanine-rich sequences were shown to contain B-type sugar puckers, which was considered as a major marker discriminating structure B from structure A (Aboul-ela et al., 1988; Benevides et al., 1986). However, the present work shows that sugar puckering is not an unambiguous indicator of DNA conformation, because B-type sugar puckering is compatible with a structure of the present duplex of

915

An Intermediate B/A Duplex of DNA Ê ) of the various double helices of d(CCCCGGGG) Table 5. Minor groove width (P-P separation minus 5.8 A Phosphate pair G8-G13 G7-G14 G6-G15 G5-G16

Average over all trajectories 6.6 9.2 9.2 6.6

(1.66) (1.85) (1.61) (1.56)

B-DNA starta

A-DNA startb

Canonical A-DNAc

Canonical B-DNAd

Crystal structuree

5.0 9.0 9.0 5.0

5.8 8.5 8.5 5.8

12.6 12.6 12.6 12.6

5.9 5.9 5.9 5.9

10.0 10.6 10.6 10.0

The standard deviations are displayed in parentheses. a Average structure over the last nanosecond of the MD trajectory starting from the canonical B-DNA (Arnott & Hukins, 1973). b Average structure over the last nanosecond of the MD trajectory starting from the canonical A-DNA (Arnott & Hukins, 1972). c Arnott & Hukins (1972). d Arnott & Hukins (1973). e Crystal structure of d(CCCCGGGG) (Haran et al., 1987).

d(CCCCGGGG), whose helical geometry is unambiguously A-like (Figures 5, 7, and 8). The molecular structure of the present intermediate or mixed B/A double helix of d(CCCCGGGG) has all eight base-pairs coupled in the WatsonCrick manner, all sugars puckered as in structure B, and the glycosidic torsion angles all fall into the anti region. These are the expected properties. The ®rst unexpected property becomes evident if one looks at the double helix along its long helical axis. Then one can see a wide central hole (Figure 5), which is entirely inconsistent with structure B, whereas the hole is characteristic for structure A

(Conner et al., 1984). The hole results from stacking the guanine ®ve-member ring on the six-member ring of the neighboring guanine base, while the cytosine bases almost fail to overlap with the neighboring bases. The second remarkable property of the intermediate B/A double helix of d(CCCCGGGG) is its minor groove, whose width increases from the duplex ends towards the duplex center. Similarly, gradual changes in the minor groove width were observed with the oligo(AnTn) sequences connected with DNA bending (Alexeev et al., 1987), but this effect was opposite, i.e. the width decreased with

Figure 8. The solvent-accessible surface representation (Connolly, 1983) of (from left to right) the canonical B (Arnott & Hukins, 1973), the present intermediate A/B, and the canonical A structures (Arnott & Hukins, 1972) of the duplex of d(CCCCGGGG). Top row, view from the major groove side; bottom row, view from the minor groove side.

916 the increasing length of the purine tract (Chuprina et al., 1991; Hud & Feigon, 1997; Hud et al., 1999). The third remarkable property of the intermediate B/A double helix of d(CCCCGGGG) resides in its central CG step. It follows from simple considerations based on the Lennard-Jones part of the empirical potentials that the negative propeller twist of the Watson-Crick pairs, which is typical for most base-pairs in the crystal structures of DNA (Dickerson, 1983), results in unacceptable clashes that can be relieved by various maneouvres (SÏponer & Kypr, 1990, 1993). This clash-avoiding behavior of the CG step is so general that its manifestations are seen here in the duplex of d(CCCCGGGG) and in the CG steps of other DNA fragments (Mauffret et al., 1989; El antri et al., 1993; Lefebvre et al., 1995a,b). The base-pairs lack the negative propeller twist in the central CG step of the present duplex of d(CCCCGGGG) and are buckled in such a way that the guanine as well as the cytosine bases, point to each other (Figure 9). This way of buckling is incompatible with the standard geometry of structure B because the guanine bases are large and hence extend across the double helix center where they would generate an unacceptable clash. The clash is avoided in the present double helix of d(CCCCGGGG) by elimination of the usual negative propeller twist and shifting the guanine bases from the helix center towards its periphery. A consequence of this shift is the appearance of the central double helix hole and other deviations from the canonical structure B.

An Intermediate B/A Duplex of DNA

Figure 9. Schematic presentation to show tilting of bases and the CG step deformation in the intermediate B/A duplex of d(CCCCGGGG). This Figure shows the minor groove widening towards the central CG step.

to-DNA world transition that had originally evolved on RNA.

Materials and Methods Conclusions Independent CD spectroscopy, NMR spectroscopy and molecular dynamics data consistently indicate that the octamer d(CCCCGGGG) associates into a DNA double helix having unprecedented properties. According to the CD spectrum shape, the global double helix architecture and the double helix central hole, it is a member of the A-DNA family of structures. However, it simultaneously contains B-type puckering of the deoxyribose rings, which probably causes the duplex to undergo the cooperative TFE-induced transition to structure A. The aqueous double helix of d(CCCCGGGG) extends our knowledge of possibilities of the DNA secondary structure. The present results simultaneously remove the contradictions in the literature regarding DNA recognition by the IIIA transcription factor because they show that B-type sugar puckering can occur in a DNA double helix whose overall architecture is more similar to structure A than structure B. The present B/A double helix of DNA potentially has a far-reaching biological signi®cance because the B-A transition of DNA accompanies both replication and transcription. Last but not least, the present intermediate B/A double helix might have helped DNA to take on functions during the RNA world-

Oligonucleotides and the NMR samples The oligonucleotides used in this work were synthesized and puri®ed as described (Kypr et al., 1996), or bought from Generi Biotech, Hradec KraÂloveÂ, Czech Republic, or Laboratory of Plant Physiology of the Faculty of Sciences of the Masaryk University in Brno. Three independent samples of d(CCCCGGGG) were analyzed and compared by electrophoresis in denaturing polyacrylamide gels on Hoefer slabs (Kovanda et al., 1996), and by UV absorption spectroscopy and CD spectroscopy. The latter measurements were performed under various conditions, including those used for the present NMR measurements, to make sure that the NMR and CD spectroscopies measured the same double helix of d(CCCCGGGG) and that this double helix was suf®ciently stable with respect to small variations in temperature, ionic strength and the pH values as well as the oligonucleotide concentration. The NMR samples used in this work contained 1.95 mM d(CCCCGGGG) strands dissolved in 10 mM sodium phosphate buffer (pH 7.4), containing 100 % 2H2O or 90 % H2O and 10 % 2H2O. CD spectroscopy The CD spectra were measured using the Jobin-Yvon Mark VI dichrograph in thermostatted (Haake cryostat) cells. The oligonucleotide samples were denatured (ten minutes at 85  C) before the CD measurements that were mostly carried out in 0.1 cm pathlength rectangular Hellma cells at 0.8 mM (nucleoside residues) oligonucleotide

917

An Intermediate B/A Duplex of DNA concentrations, giving about 0.7 absorbance unit at 260 nm, which gives the optimum signal-to-noise ratio. The sample used for NMR measurements was ®rst measured in a Hellma 0.01 cm pathlength sandwich cell to determine that its CD spectrum was essentially the same as that of the diluted sample measured in the 0.1 cm pathlength cells. The NMR sample of d(CCCCGGGG) was more thermostable, which is natural because the duplex is more populated if the oligonucleotide concentration is higher by an order of magnitude. The B-A transition was induced by adding cool 100 % TFE (Sigma) to 1 mM d(CCCCGGGG) dissolved in 10 mM sodium-phosphate, 0.3 mM EDTA, pH 7, at 0  C. The ion and d(CCCCGGGG) concentrations were decreased by the additions of TFE. Starting from 58 % TFE, the measurements were performed in 0.2 cm pathlength cells. Elliplicity is given in units of Mÿ1 cmÿ1, whereas the concentration is in terms of the nucleoside residues of the oligonucleotide. The oligonucleotide concentrations were determined from the UV absorption spectra (Unicam 5625 UV spectrometer) of the denatured (temperature 90  C) oligonucleotides dissolved in 1 mM sodium phosphate, 0.3 mM EDTA, pH 7, using the extinction coef®cient of 9150, 9230 and 8870 Mÿ1 cmÿ1 at 260 nm with d(C4G4), d(C2G2) and d(CG)4, respectively. These values were calculated according to Gray et al. (1995). NMR spectroscopy The NMR experiments were performed on a Bruker AVANCE 500 MHz spectrometer equipped with a z-gradient triple resonance 1H/13C/BB probehead. The data were processed on Silicon Graphics computers (Octane, O2) with Bruker NMRSuite programs. For assignment purposes, the following spectra were acquired: 2D NOE spectra (Jeener et al., 1979) in 2H2O at 20  C with mixing times ranging from 50 to 200 ms; 2D NOE spectra in H2O at 5  C using WATERGATE solvent suppression (Piotto et al., 1992) with a mixing time of 150 ms; TOCSY spectra (Braunschweiler & Ernst, 1983) with MLEV-17 mixing (Bax & Davis, 1985) at 20  C and the mixing times ranging from 20 to 140 ms; the heteronuclear 1H-13C natural abundance HSQC spectrum (Bodenhausen & Ruben, 1980); and the 1H-31P correlation spectrum (SklenaÂrÏ et al., 1986). All 2D NMR spectra were collected with the StatesTPPI quadrature detection in t1 (Marion et al., 1989). The recycle delay of 2.2 s was employed in all measurements. The 1H, 13C, and 31P signals were referenced indirectly in all 2D experiments (Wishart et al., 1995). For the 31P resonances, the ratio (31P/1H) ˆ 0.404808688 was used at 500 MHz (R. Fiala, personal communication). Three-bond proton-proton coupling constants of the deoxyribose residues were obtained from a DQF-COSY spectrum (Piantini et al., 1982). The acquisition parameters were 2048 points in t2, 1024 points in t1, and 32 scans per t1 block. The spectrum was zero-®lled to 2048 real points in both dimensions. Three-bond and fourbond proton-phosphorus coupling constants were extracted from a 2D J-resolved 1H-31P correlation spectrum (SklenaÂrÏ & Bax, 1987). The acquisition parameters were the spectral widths 2500 Hz (t2), 202 Hz (t1), 1024 complex points in t2, 100 complex points in t1 and 192 scans per t1 block. The spectrum was zero-®lled to 1024 and 2048 real points in t1(31P) and t2(1H) frequency dimensions, respectively. A 2D HSQC spectrum acquired

with very high resolution (Schmieder et al., 1992), was used to extract the three-bond carbon-phosphorus coupling constants. The acquisition parameters were the spectral widths 4000 Hz (t2), 3800 Hz (t1), 2048 complex points in t2, 780 complex points in t1, and 160 scans per t1 block. The spectrum was zero-®lled to 2048 real points in both dimensions. Distance restraints Distance restraints for the non-exchangeable and exchangeable protons were obtained from the 2D NOE spectra measured with tm ˆ 150 ms at 20  C in 2H2O, and at 5  C in H2O. The data were collected with sweep width of 5000 Hz (non-exchangeable protons) and 7500 Hz (exchangeable protons) using 1024 and 2048 complex points in the t1 and t2 dimensions, respectively; 32 scans were collected per t1 increment. Prior to Fourier transformation, the t1 and t2 dimensions were zero-®lled to 1024 and 2048 real points, respectively. Cross-peaks were integrated using Aurelia software package (Neidig et al., 1995). All distances were calibrated using the strongest resolved H5-H6 cross-peak set to 0.245 nm. Endocyclic torsion angles, sugar pucker parameters The concept of pseudorotation was used to describe the conformational behavior of the deoxyribose rings (Altona & Sundaralingam, 1972). Their pucker parameters were related to the scalar coupling constants (3JH10 -H20 , 3JH10 -H200 , 3JH20 -H30 and 3JH200 -H30 ) using the appropriate Karplus equations as implemented in program PSEUROT (de Leeuw & Altona, 1983) using the twostate model. The scalar coupling constants were determined by quantitative line-shape simulations of the cross-peaks of the 2D DQF-COSY spectra using program CHEOPS (Schultze & Feigon, unpublished program). In addition, reliability of the sugar pucker parameters was checked using the 1H and 13C chemical shifts (GiessnerPrettre & Pullman, 1987; Dejaegere & Case, 1998), TOCSY peak intensities (Remenowski et al., 1989) and NOE data analysis (Wijmenga et al., 1993). Exocyclic torsion angles a, b, g, z, e, w Restraints for the torsion angles a and z, were derived from a temperature-dependence of the 31P chemical shifts (Gorenstein et al., 1982; Schroeder et al., 1989; Karslake et al., 1990; Nikonowicz & Gorenstein., 1990; Roongta et al., 1990; El antri et al., 1993; Gorenstein, 1994). One-dimensional 31P spectra were obtained in 2 H2O for temperatures ranging from 20  C to 80  C. Each spectrum was acquired with 4096 complex points, 128 scans, and the spectral width of 1000 Hz, and apodized with 60  phase-shifted square sine bell function. The 31P chemical shifts were referenced indirectly to TMP in the 1D measurements. The b and e restraints were derived from the 1H-31P and 13C-31P heteronuclear scalar coupling constants (Wijmenga & Buuren, 1998). Conformationally relevant 1 H-31P scalar couplings were determined by quantitative line-shape simulation of the H30 -P and H40 -P cross-peaks in the 2D J-resolved 1H-31P correlated spectrum using the program CHEOPS. The 13C-31P J-couplings were estimated from signal splitting of the H40 -C40 and H20 -C20 cross-peaks in the 2D HSQC spectra (Schmieder et al., 1992). The torsion angles b and e were determined using

918 the appropriate Karplus equations (Wijmenga & Buuren, 1998). Three independent sources were used to obtain the information about a conformation of the g torsion angle. Where it was possible the g torsion angle was calculated from the H40 -H50 and H40 -H500 three-bond scalar coupling constants (Wijmenga & Buuren, 1998). These scalar coupling constants were determined by quantitative lineshape simulation of the H40 -P5 signals in the 2D 1H-31P J-correlated spectrum by the program CHEOPS. The H50 -H500 coupling constant was frozen to 12.1 Hz during the CHEOPS simulation. Additional information was extracted from 40 ms mixing time TOCSY spectra (Kolk et al., 1998) and from the chemical shift of the C30 , H20 and H200 resonances (Dejaegere & Case, 1998). The restraints for the torsion angle w were assessed using both NOE distances of the H8/6-H10 ,H8/6-H20 , H8/6-H200 , H8/6-H30 proton couples (Wijmenga et al., 1993) and the proton chemical shifts (H10 ,H20 ) (Wijmenga et al., 1997). The NMR data only permit us to specify conformational regions of the a, g, and z torsion angles. Boundaries of these regions were taken from the oligonucleotide crystal structures (Schneider et al., 1996; Gelbin et al., 1996). Molecular dynamics methods and simulation protocols The starting canonical structure A (Arnott & Hukins, 1972) and structure B (Arnott & Hukins, 1973) of d(CCCCGGGG) were generated using the NUCGEN module of AMBER 5.0 (Pearlman et al., 1995; Case et al., 1997). The starting coordinates were also obtained from the crystal structure of d(CCCCGGGG) (NDB code ADH012; Haran et al., 1987). The structures were neutralized by sodium cations initially placed using the Coulombic potential terms with the LEaP module of AMBER 5.0. Then the neutralized structures were surrounded by a periodic box of water molecules described by the TIP3P potential (Jorgensen et al., 1983; Jorgensen, 1981). Ê away The water box was extended to a distance of 12 A from any solute atom. This yielded about 4400, 4100, and 4400 water molecules used for the solvation of the canonical A, canonical B, and the crystal structures of d(CCCCGGGG), respectively. The all-atom force-®eld parameters described by Cornell et al. (1995) were used in the simulations. All calculations were carried out using the SANDER module of AMBER 5.0, with SHAKE (Ryckaert et al., 1977) on the hydrogen atoms, with a tolÊ , a 2 fs time-step for the Newton's erance of 5  10ÿ4 A equations, temperature coupling (Berendsen et al., 1984) Ê cutoff applied to with a time constant of 0.2 ps, a 9 A the Lennard-Jones interactions, and the constant pressure of 1 atm. The non-bonded pair list was updated every ten steps. Equilibration was started by 1000 minimization steps with the position of the DNA ®xed. After this initial step of the equilibration protocol, all subsequent simulations were performed using the particle mesh Ewald (PME, Essmann et al., 1995) approach for evaluating long-range electrostatic effects. The PME charge grid Ê , and the charge grid was spacing was approximately 1 A interpolated using a cubic B-spline with the direct sum Ê direct space cutoff. Next tolerance of 10ÿ6 at the 9 A steps of the equilibration protocol were the following: 25 ps of MD simulation with the position of the DNA Ê 2) ®xed, 1000 steps of minimization with 25 kcal/(mol A restraints placed on all solute atoms, 3 ps of MD simuÊ 2) restraints placed on all lation with 25 kcal/(mol A

An Intermediate B/A Duplex of DNA solute atoms, and ®ve rounds of 1000-steps minimization where the solute restraints were reduced by 5 kcal/(mol Ê 2) during each round. At the end of the equilibration A protocol, a 20 ps MD simulation was carried out with the system heated from 100 to 300 K over 2 ps. In the course of our MD simulations, the coordinates were saved after each picosecond. Calculations of helical parameters The Dials and Windows (Ravishanker et al., 1989) interface to Curves (Lavery & Sklenar, 1988) has been used to determine the helical parameters (Dickerson, 1989). Pseudorotation phase angles and amplitudes were calculated using the Altona & Sundaralingam (1972) convention. Denotation Nucleic acid residue names are referred to as oneletter codes in the text. Where necessary, a subscript for the residue number is also presented. The residue number increases in the 50 to 30 direction, with the ®rst and second strand numbered 1-8 and 9-16, respectively, in the MD simulations. Both strands are identical in the NMR measurements and their residues are numbered 1-8 in the 50 to 30 direction. The conformational regions were denoted according the Klyne-Prelog convention (Markley et al., 1998).

Acknowledgments This work was supported by grants A4004701 from the Grant Agency of the Academy of Sciences of the Czech Republic and grant VS 96095 from the Ministry of Education of the Czech Republic. The authors thank the Academic Supercomputer Center in Brno and Prague for providing access to computer facilities.

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Edited by I. Tinoco (Received 11 November 1999; received in revised form 9 February 2000; accepted 9 February 2000)