J. Mol. Biol. (1992) 226, 775794

Conformational

Sub-states in B-DNA

Marc Poncin, Brigitte Hartmann

and Richard Lavery

Laboratoire de Biochimie The’orique Institut de Biologic Physico-Chimique 13 rue Pierre et Marie Curie Paris 75005, France (Received 12 August 1991; accepted 9 Ma,rch 1992) Theoretical studies of the sequence-dependent conformation of B-DNA have been carried out using Jumna. a helicoidal co-ordinate minimization algorithm. The results obtained for a series of six oligomers with repetitive sequences show that, with the exception of the homopolymers (dA); (dT), and (dG); (dC),, all sequences can adopt a variety of conformations characterized by considerable changes in helicoidal parameters and also in sugar puckers which adopt C&- endo (falling into 2 classes) or, in the case of pyrimidine nucleotides, Ocl,)- endo forms. These studies lead to an improved understanding of t,he role of base sequence on DNA conformation and point to a number of interesting correlations between the various structural parameters describing the double helix. Kryurordn:

DNA;

conformation:

base sequence:

1. Introduction

molecular

modelling;

fine structure

stretching) available within Jumna to ensure the stability of each local energy minimum. These possibilities combined with a detailed conformational analysis using the Curves algorithm (Lavery & Sklenar, 1988,1989), enable us to take a step beyond previous modelling of DNA fine structure. We have thus undertaken a systematic study of symmetric DNA oligomers of different sequences in order to try and understand the mechanics that govern the fine structure of DNA and the correlations between different structural parameters. These data, once assembled and analysed, should enable us t.o generate a set of rules capable of predicting conformation starting only from sequence data. This article presents the first step t,owards our goal and deals with homopolymeric and alternating sequences of B-DEA. By studying six repeating and hence helically symmetric sequences, (CG),. (TA),, (TO),. (GA),,, (GG), and (AA),, we are able to generate conformations for the ten unique dinucleot.ide sequences and for eight of the 32 unique trinucleotide sequences. The fact that we have been able to generate a variety of stable conformations for eac>h chosen oligomeric sequence also means that we are able to judge to what extent these dinucleotides or trinucleotides determine the local structural parameters of the double helix. Future work will involve studies on longer sequence repeats, which will enable us to investigate all trinucleotide and tetranucleotide sequences and will also address the

It is clearly necessary to understand the sequence-dependent fine structure of DNA if we are to be able to understand its biological behaviour and, in particular, the recognition of specific DNA sites by proteins or drugs. Difficulties in obtaining appropriate crystals of oligomeric B-DNA and the incomplete nature of nuclear magnetic resonance spectroscopy data for DNA oligomers hinder the accumulation of experimental data in this field. It thus seems that theoretical modelling may play an important role. if the modelling techniques employed are suficiently realistic. Over the last few years, we have developed an energy optimization procedure, termed Jumna (Lavery, 1988). particularly adapted to the study of nucleic acids and having several advantages over classical molecular mechanics techniques. In particular, the fact that Jumna functions directly in a helicoidal parameter system enables us to easily impose symmetry on a DNA segment and thus to reduce the number of variables representing the conformation by roughly two orders of magnitude. This. in turn, reduces the risk of getting trapped in local energy minima and gives us the possibility of making a more thorough search of the conformational space corresponding to any chosen allomorphic form of DKA. This search is also assisted by using forced helicoidal deformations (twisting and 775 1)~2-283fi/92/15077520

$03.00/O

0

1992

Academic

Press

Limited

question of the sequence-dependerlt tiexibility of I>?;A. Although a complete picture of I)r\;A fine st.rucat,ure almost’ certainly requires studies at least,at the tetranucleotide level (Calladine. 1982; Yanagi it al.. 1991). the present, work already brings to light a number of interesting findings concerning t,he sequence-dependent and the sequence-independent nature of DNA conformation. One import’ant result. which is already clear at, this level, is that H-DNA is not a single conformation. but rather an ensemble of distinct, conformational sub-states. Finally, we are able to show t’hat our approach to modelling DNA is supported by many features of the comput’ed structures, which are found to be in good correlation

with

experimental

knowledge.

1Z’r

would hope that these data should thus be of interest from a theoretical point of view and as a means of improving the refinement of experimental

data on l>KA uniformly

require

structure molecular

which,

today.

almost

modelling.

2. Methodology The calculations presented were all performed using the Jumna (Junction Minimization of Nucleic Acids) algorithm. which has been the subject of a number of previous publications (Lavery. 1988; Sun rt al.. 1988; Ramstein Br I,avery. 1988: Hartmann et al.. 1989). ,Jumna models DN.4 flexibility by a combination of helicoidal parameters describing the position of each nucleotide (3’-monophosphate) with respect to a common helical axis system. single bond rotations at the glycosidic link and within tht phosphodiester backbone and valence angles within the sugar rings. All other valence angles and all bond lengths are taken to be fixed. The independent variables of each nucleotide are consequently 3 translations and 3 rotations, which position the nucleotide wit,h respect to the helical axis system. the glvcosidic dihedral. 3 valence angles and 2 dihedrafs within the sugar moiety and 2 backbone dihedrals &(C,,,-Co,,-O,,,,~P) and I(C,,,,~O,,,,~P~O,,,,). Other sugar and backbone variables are dependent and are determined by the elosurr conditions that involve the C,,,, -(I,,,, bond length within each sugar ring, the internucfeot’idr bond and the valence angles P~Oo,,+‘(,,, and qs,,-qsq C)o,,-Co,,-(I,,,,. These constraints are imposed t;in harmonic energy penalty terms. The corresponding force constants were adlusted to satisfy closure distances to within 0.02 A (1 A = 0.01 nm) and closure angles to within 1’. Full energy derivatives with respect to the independent, variables are calculated. st’arting from analytic atomic forces and minimizations are caarried out using a conjugate gradient algorithm. The force field employed is termed Flex (Lavery rl nl., 1986a,b). which uses the following conformational energy expression formed from a series of pairwisr additive terms: E = ZQiQj/&(R)Rij + C( - A,,/R; + Bij/R,!,‘) +X[cos t?(- A;B/R;+ ByjR;j2, + (1 -cos @( - A,,/R; + Bij/R;j’)] + c v,/q 1 * cos ‘VSTS)+ Z Va(CJa- C&y. The first term of this formula is the electrostatic energy, calculated as a sum of interactions between atomic mono-

poles Q, darnfbrcl by a tlif+~$ric~ t’unc~1roli ::(li;. wlrl~~l~ is described below. Thr monopoles WV employ are’ (~af~~LIlat,rtl by the Hiic,krl-Del Itr method. specially rf:f)ar.arnrtrrizt~cl to obtain t,he best possible tit with ~1) initio rlrc+ro st,atic potential and field distributions around thts nu(.leil, arid subunits (I,avr*ry P( (I/.. 19X4). Th(, n(‘xt :1 tcsr’n~; represent thr Lennard m,Jonrs OI tlispersiofl-rPfrrllsioti energy calculated with a O-f 2 dependrnc~r and using. iti part,, the parameter set drvt~loprd hy the group of I’olttA\ (Zhurkitl uf al.. 1980). Hydrogrll bonds arr dralt lvith I,> t,he lattrr 2 of these tcxrms. whictl take into itc,lwtrttt angular dependenc>r. tit,t& to cfuant,um c,hrrnicA ~~af(~ulaCons. by mixing togrthrr 2 sets of .-f.H parameters using a c>osinr func,tion of thtl angle f’ormed by the Eecator’s N-H and H-Y f’or a tmrd ?i H. .\‘. All of thrsrs tcsrttls at’. helical symmrtry by simply rrmoving all but, I of racah set of symmetry-equivalent variahfrs: this leads to a furt,het important reducation in the dirnensionafity ot’ thtl c~nt~rg~ hypersurfacxr to be studied and c*onsequrnt.ly simplifies t htl search for locaal minima. It should also be nottktl that a c~ombinatioli of internal and tirfic.oidaf variaf)ff~s is a “natural“ cti0ic.r for studying I)?;.4 tleforn~ation that. in our rxprriencae. leads to many fewer f)roblrms of local minima than are rnc7)ulrterrcl with (‘artesian variable minimization. This formulation also enables us to set up a means of verifying the “stability” of any loyal minima that art’ found. The 1st step in this procedure involves using all the minimal energy conformations located with a given base sequence as starting pointZs for all other bast: sequences investigated. This “crossing-over” is cxont,inurd until sub sequent energy minimization leads to no new caonformathe stable tions. The 2nd step involves deforming conformations with respect to cahosen helicoidal variables

Conformational

to ensure the existence of a locally quadratic energy surface. The variables currently used for this purpose are the total rise and twist of the double helix. Deformations are performed by imposing an harmonic constraint on the total value of the corresponding variable (which leads t’o energy gradients on all individual base-pair rises or twists) and t,hen carrying out an energy minimization leaving all other variables free to vary. The deformation energy curves present.ed in this article thus correspond to adiabatic energy mapping of overall rise or twist. Tt is lastly remarked that when symmetry is imposed on a DXA oligomer. it is possible to avoid end effects and to gain time by clalculating only the energy of the central repeating unit (flMu. where M = N/2 for an oligomer formed from ;V repeating units) plus the int’eraction rnergy of this unit wit,h all the other units wit.hin the oligomer (EM,). This enablesus to calculate t)hequantity EM. as defined btalow. which corresponds to the energy of 1 repeating unit, within the environment of an effectirel) infinite DRA: IT, or.

alternatively.

= 1/L!?,,+ by

noting

1/2&=,,JEM, that

EMWI..,

= E,W,,+,:

All minimizations carried out here were made in terms of Ii’,M for the central nucleotide pair (mononucleotide symmetry) or dinucleotide pair (dinucleotide symmetry) of the oligomers studied. The total energies of the oligomers listed in the following section are obtained by simply summing the EM values (M = 1. S) for all the repeat’ing units in each oligomer. Calculations were carried out for the 6 oligomers listed below (referred to hereinafter by the 2-letter sequence c,orresponding to the 1 st nucleotide pair): (‘G: TA: TO : (iA: t:u: A.4.

in B-DX;1

&b-states

tY:CGC!(:CGCGCGCG TATATATATATATA TGTGTGTGTGTGTG (:AGA(;AUAGAGA(=A GGGGGGGGGGGGGGG .-LUAAAAAAAAAAAA

Togrt,hrr. thrsr oligomrrs contain thtx 10 unique tlinucleotide sequences and 8 of t,he 32 unique trinucleotide sequences. Energy optimizations on the 1st 1 oligomers (c>onsisting of 14 base-pairs) were performed under dinucrleotide symmetry constraints and the latter 2 oligomers (consisting of 15 base-pairs) were constrained to mononucleotidr symmetry. In addition, int.erstrand dyad svmmrtry (homonomous constraint.) was imposed on t.hr (‘G and TA oiigomers. Initial optimizations used R-D?;4 fibre po-ordinates as starting points (Arnott et al.. 1980 and S. Arnott. personal communication), however. as mentioned above. subsequent calculations were performed by using all the stable conformations obtained as new start.ing points (switching base sequences). The resulting conformations can thus be considered as independent of their starting points. Moreover. each minimum rnergb st~ructurc~ was also subjected to stretchingcompression and twist,ing-untwisting deformations. also tlescribrct above, to pnsure its stability over a range of helicoidal paramtxters:. Wr have thus taken considerable cani in ensuring that each conformation presented is well c.haracterized. hlajor conformational changes leading away from the c-anonical H form (Srinivasan & Olson. 1987) werr not the subject of the current study and their nature will he inrest’igat’ed in future work. Minor conformational changes leading to other minimal energy conformations than those detected vannot hp rxcluded. but

777

given the procedures employed it is unlikrly that, many such stat.es could exist. The conformational parameters used to describe the minimal energy conformations follow the Cambridge convention for DNA (Dickerson et al.. 1989) and were calculated using the Curves algorithm (Lavrry & Sklenar. 1988. 1989).

3. Results The energy optimizations described in Methodology led to a total of 20 conformations; three, four, six and five conformations for the dinucleotide sequencesCG, TA, TG and GA, respectively, and a unique conformation for each of t,he homopolgmeric sequencesGG and AA. The energies of these minima are presented in Table 1 and are hereinafter referred to by the name of the central nucleotide and a number indicating the order of decreasing stability shown in the Ta,ble, e.g. TG,, TG,. .. TG,. The full structural descriptions of each conformation, including helicoidal parameters, backbone angles, sugar puckers and groove geometries (Stofer & Lavery: unpublished results) are present’ed in the Appendix. The first and most striking point to note from these results is t,hat, with the exception of the homopolymeric sequences,each oligomer can adopt a number of distinct conformations in terms of both helicoidal parameters and sugar puckers with only minor changes in internal energy. This does not imply. however. the existence of a continuity of conformational st.ates around a single canonical structure. Each of the minimal energy conformations described has been shown to be stable over a range of twist and stretch deformations and no further minima were located at energies close t,o those presented. We will present the conformational features of each of the structures obtained and attempt to show how the existence of these strut!tures can be rationalized.

(a) Sugar

p,uckw.s

Table 1 indicates that the most stable conformat,ions are expect,ed

obtained with in R-DNA

Co,,-sndo

sugar

puckers

as

(see also the Appendix for details). However, Oc,,,-endo sugars can also be introduced for pyrimidine nucleotides with only slight decreases in stability and, in one c‘ase,can even lead to a more stable conformation (TGd). It is remarked that both crystallographic and n.m.r.t studies

of

oligomers

confirm

thr

existence

of

puckering (Metzler et al.. 1990: Searle & 1990: Privk et nl.. 1991: 8chmitz et rrl.. It should be noted that n.m.r. spet’troscopy

(ICI,,-endo

Wakelin, 1991). combining both (Jf detcbrmining

COSY and NOESY the phase angle of

data is capable thr sngar rings

t Abbreviations used: n.m.r.. nuclear magnetic8 resonance spect,roscopy; COSY. 2-dimensional correlation spectroscopy: BOESY. P-dimensional Overhauser effect spectroscopy.

nuclear

Table Total

enrrgies

(kcallmol)

conformations

1 qf

the

oliyomers

of the 6

((‘(:)I4

-821.3

-8lX.i

PA),,

- 562.1

- 5.59.i

-8160 ** - 5.56~3

(‘lx),,

- 692.7

-692.2

-684.8

W),,

-686.9

-678.8 *

-676.1 *

(AA),, (M;),5

~ 603.1 - 873.8

minimal studied

- 553.1 ** -691.5 ** -6758 *

-695.1 * -670.6 *

r~t.rrqy

-687~0 * + -

30

An asterisk (*) indicates the presence O,,,,-endo sugar puckers: 1 cal = 4.184

of 1 or 2 nucleotidrs

’

’

I

I

I

I

100

I I 150

I

I

with

J.

with good precision, as detailed studies have shown (Gochin &, James, 1990; Schmitz at al.. 1990). (I(,,,-endo puckers for pyrimidine nucleotides have also been observed in theoretical st’udies (Zhurkin et al., 1990). For sequences containing both cytosine and thymine, it is found t#hat cytidine can change t,o O,,,,-endo puckers somewhat more easily than thymidine. Otl,)- endo sugars are found with a range of phase angles (P) from 82” to 92 ” and amplitudes (il ) between 32” and 39”. It should be noted, however, that none of the conformations we have generated contains homopyrimidine strands with uniquely (ICI,,-endo puckers. Conformations with OC,,,-endo puckers always have such sugars sandwiched between two CC,,,-endo sugars. Tf we look in more detail at the CC,,,-endo puckers. different families can be distinguished. For purines. CC,,,-endo sugars lie in the range P = 166” to 185” wit’h A = 34” to 3X”> while a second group close to the C exo class P = 145” to 160” is associated with (ixigher amplitudes A = 41” t,o 45”. Pyrimidines. in addition to the O(,,,-endo puckers mentioned above. can also adopt two classes of south puckers: P = 150” to 165” with d = 34” to 38’ or P = 145” to 160” with A = 42’ to 46”. Note that’, while the second class is almost identical with that of the purine sugars, the first class has smaller phase angles than those found wit,h purines and is thus less well separated from the lower phase class. This is in agreement with experimental findings (Uochin & ?James! 1990). These results are illust,rat,ed by Figure 1. To simplify further discussion, we introduce a one-letter code to represent each of t’he three classes of sugar pucker observed: 8 (Sout)h high phase, low amplitude C’c,,,-endo); X (eXo, Ion phase. high amplitude CC2,,-endo close to the Cc,,)-exe conformation); and E (East low amplitude O,,,,-endo). (b) Helicoidal

0

parameters

The data describing the helicoidal parameters associated with each conformation are collected and presented as histograms (Figs 2 and 3), which made. enable rapid comparisons to be

Phase

(deg

1

Figure 1. sugar phase as a function of amplitude for the 20 optimal conformations of‘ the 6 oligomers (+. S high phase, low amplibde (‘,z,,-rndo; x. X low phase. high amplitude (‘,,,,-rndo; o, E low amplitude O,,,,-undo)

Inter-base-pair parameters for the ten unique dinucleotides (CG, TA, TG, GC, AT, GT, GA; AG. GG and AA) are arranged in the classes: Pyr-Pm. Pur-Pyr, Pm-Pur (Fig. 2). Base-pair axis and intrabase-pair parameters are arranged by trinucleotide sequences. In this case, only eight of the 32 unique trinucleotides feature in the oligomers presently studied: CGC, TAT, GTG, TGT. AGA, (:A(:, 6(X: and AAA. These trinucleotides belong to eit’her the Pyr-Pur-Pyr or the Pur-Pur-Pur classes and have been grouped following the oligomer sequence from which they are obtained (Fig. 3). Xote that t,hr translational intra-base-pair paramet,ers three shear, stretch and stagger and the rotational parameter opening are not shown in Figure 3 as they are close to zero for all t’he conformations obtained (see Table 2). The horizontal axes of the histograms in Figures 2 and 3 correspond to mean value of asso ciated parameters (see Table 2). This implies t’hat parameters that fall very close to the mean are not visible on the histograms. Each entry is also shaded to show the type of sugar puckers associated with the dinucleotide step (Fig. 2) or the trinucleotidc step (Fig. 3): black; Co,,-endo: grey; a single Ot,,,-endo sugar; white, two or three O(l,,-rndo sugars. Finally, the order of the bars for each parameter corresponds to t,he order of the strucatures given in Table 1. We will begin by discussing dinucleotide parameters. One can see that all these parameters cover wide ranges; however. their mean values are close to those of canonical R-DKA (Table 2). The largest variations are found in the case of shift, slide. rise and twist, and it can be noted that none of these parameters is determined by the dinucleotide step alone, since important variations are observed within each group of peaks (i.e. different conformatioDns of the same oligomer). Rise covers a range of 1 A and is largest for the homopolymeric (:

Conformational

Sub-states in H-D&VA

779 _---

15’

t

I

CG

TG

TG

GC

AT

g o.:!* ], 07

GT

GA

AG

-0.5}

GG AA (

,, ]] ,]

,GcG

1 g:?f

-0.5

CGC

ATA

TAT

GTG

TGT

AGA

GAG GGG AAil

TGT

AGA

GAG GGGAAA,

--~------GTG TGT

AGA

GAG GGG AAA’

1

-3.

-!t i1.

-3.5. I--

CG

TA

TG

GC

AT

GT

GA

AG

GG AA /

I’

I 0.5

1 5

0.25

0 :: F -0.25 -0.5

F 9 g z 0 ‘c

-lot -15 t

,-__~-~ si 3CG -A

_TG

GC

AT

GT

’

~~ ~-IO

&CG

TA

TG

GC

AT

GT

GA

TG

GC

AT

GT

GA

AG

GG AA ’

45. -

-

-~

-. _____

GCG CGC ATA

TAT

4

,

GCG CGC ATA

TAT

GTG

TGT

&;A

SAG (GGGAAA I

‘5

I

KG

TAT

GTG

TGT

AGA

GAG 4

GA

CGC ATA

I

42.5.

Yir 2 40. f 37.5. F

35. 32.5.

1'

30.

Figure 2. Histograms of t,he helicoidal inter-base-pair parameters (translational parameters in A. rotational parameters in deg.. pale shading indicates the presence of (I,,,,-endo sugars. a diagram indicating the positive sense of each parameter is also shown with the dpad axis pointing towards the minor groove and the 5’ to 3’ strand pointing upwards on the left,-hand side).

sequence. Twist is also very variable (29” to 44”). The highest twists are found for the Pyr-Pm steps TpG and TpA but’, as for rise, most dinucleotides exhibit a range of values above and below the mean.

Figure 3. Hi&ograms of the helicoidal base-pair axis an< intra-base-pair parameters (translational parameters in A. rotational parameters in deg.. pale shading indicates the presence of Oc,,,-endo sugars).

In alternating sequences, high twists are always followed by low twists and vice uersa as has been found in both theoretical and n.m.r. studies (Hao & Olson, 1989: Schmitz et al., 1991). Slide is also variable and shows a clear correlation with twist. The colouring of the histograms also shows that sugar puckers are clearly related to these t,hree

09 05 I 04 14,!4 I I.3 3.x

- 0.1 0.1 wo - 0.9 - 64 (NJ

t .Arnott & Hukirx (I 973). : SW Arnott rt rd. (1.980). # Excluding the (AT), confirmations. Ibartiwlarly large negatiw r&p from -20

04) O-O O-0 o-o 37

-4-l

0~0 Il.0 -0-I I).(1 - 13.3 IHI

which exhibit 23 to - 3% A.

a

I)arameters. \2:e will ret,urn to this point in section (e). I)rlo\v. Shift and tilt parameters are zero by definition in the homonomous structures ((Xi), and (TX), (homonomous meaning obeying the same rules. in this case indicating that both strands obeq’ the same rules ot s>-mm&r?, since Pyr-Pur alt,ernat,ing seyuences lead to double helives with true dyad symmetry. invornpatible wit’h internucleotide pair shift or tilt). For the remaining oligomers. sucwssive parameters have the sa,me magnitude. hut alt,ernating signs. fiarge shifts are found for several dinucteotides but OIIW more. each dinucfeotide displays a range of values. Tilts, in contrast. are almost uniquely determined b?, the dinucleotide sequenw. Ilarge positive 1ul1s (opening tow-ards the minor groove) are found c)nl~- for TpA and TpG dinucleotides, while related negative rolls occur for ApT and C;IpT. The signs ot these rolls are also uniquely determined by thv clinucteotide seyuence. Turning to base-pair axis and intra-base-pair parameters, the histogra.ms in Figure 3 again shou irnportant variations. despite the fact that the mean \Talues are again close to t,hose of the canonical IZ form. The one exception to this rule involves the more negative values of xdisp which are closer to the value found in earlier fibre diffraction studies (Arnott & Hukins, 1973). In addition. the oligomer (TA), has unusually large xdisp combined with positive inclination and thus presents some charac-

The dihedral angles describing t,he })tlosI)ho(lirstt~, backbones are give11 in Figure 5 for rac*h tlinuc~leotide seqwnc~. Sate that) sinw \vv are now wncrrned with individual bac~khow wnformations. all tfi possible dinucteotides must be listed (e.g. the ba,ckboric linkage of -4p.4 is not idrntival Lvith that of TpT). Tn Figure 1. the rang:11 of’ each dihedral is indicated 1)~ a horizontal bar and the mean valuth is marked I),v a tlot. These results are given numeric.alty in Table 3. whew it van iw seen that t ht. values obtainecl t)>, simulation i11’(1 (~Iow to 1hew seen c.r?-stallo~rap)~ic~all~ ((:rwskowiak d ni.. 19!)1 ). bnt diffrr cwnsitirrabl~~ from cawlier tibw wsuttjs (..4rnott rt al.. 1980). Figure 1 shows that, the bwkbone tlihedrals fall into three groups in terms of their variability within

Conformational

I CPG TP* TPG CPA GPC APT GPT *PC GP* *PC

GPG *PA TPC CPT CPC TPT

Alpha / /

Sub-states

in B-DLVA

781

Beta 1

TI

/ c

-

CPG TP* 50

TP~ CPA

100

150

200

50

100

150

200

50

Phase

(deg )

100

150

200

100

150

200

Phase

13eg )

GPC APT GPT *PC GP* *PC

GPG *PA TPC CPT CPC TPT

I

I

I

I

-200-180-160-1

IL

1 1

50

-140-120-100-E

Figure 4. Variation of the backbone dihedrals for the optimized oligomer conformations (deg.). The range of each parameter is indicated by a horizontal bar and its mean value by a dot.

the minimal energy conformations detect’ed: CLand y vary very little, fl and E have a range of about 20” and [ is verv variable with a range of roughly 50”. These variations are also in line wit,h experimental findings (Saenger, 1984; Roongta et al., 1990: Kaluarachchi et al., 1991) and lie well within the ranges defined by energy minimization on doublestranded dinucleoside monophosphates (Srinivasan & Olson. 1987). Another general remark that can be made is that rather small variations in backbone dihedrals can lead to large changes in helicoidal parameters. Figure 5 shows t.he striking influence of sugar pucker on backbone dihedrals. This clearly illustrates t’hat a phosphodiest,er junction bounded by

-16OlLldUhJ 50 -80

loo

150

-160 200

50

I,,,I~IHI~ 100 150 Phase

b’“’

200

(deg 1

Table 3 Mean backbono parameters sirn u&ions compared with Min

- 73 16-l 50 9-i

;; ‘r 6 E ;

- 14”

x Phase

- 150 82

-Iii

from the present experimental results

Max.

Mean

-59 186 63 149 -Iti2 - 90

-66 176

-98 1x4

t See Grzekowiak rl nl. (1991) $ SW Arnott Pf al. (1980).

57 134 -172 -117 -117 151

Decnmert -65 167 51 I19 - 157 - I20 - 10s 146

B80$ -41 13.5 37 139 -134

-157 - 102 154

Phase

(deg i

Figure 5. Bstributions of backbone dihedral angles and of the glycosidic angle (deg.) as a function of sugar puckering (the symbols + , x and o indivatp sugar pucker groups and are defined as for Fig. 1).

an ()(lr) -endo greatly dihedrals the mean dihedral associated

sugar on the 3’ or on the 5’ side leads to reduced variability in all the backbone and. in some case. to dist.inct, changes in values. This is true also for the glycosidic x. which is found to be more flexible when with (I,,,, -endo sugars (Fig. 5). It also

-I Propeller

C(,,p7dO car :-0.92 IO

(deg 1

Slide

sugars (2Opt) ‘0

++ . + :,* * t _ *

Let0 (&!q)

sugars (20 ptl

7 I I , I 11Lc -,20 -I’)0

Zeta (3’ side)

ciT,-endo

silqars

COTz-0.76

(52 pi)

-140

-100

ideq 1

+ + * :

?I a 6

0

: t* I * ** +I

-IO

* . r--i

-20

-140 / I 1I 460 -140

(deg 1

c(pp5mYo car z -0.96

*

0

0

”

’ 1 ’ 1’ ‘J 3 3.5 Rise (deg 1

/

-10

* I

4

,i -IO

-5

0

5

301

IO

Roll (deg I

Figure 6. C:orrelat,ions between helicoidal parameters (thr correlation coefficient is indicat)ed at the top of each diagram with the number of data point,s used).

adopts distinct mean values following the sugar pucker families described above: - 110” for phase angles of 160” to 185”. - 114” for phase angles of 140” to 160” and -142” for phase angles of 80” to 95”. The associated ranges of x in each family a,re 16". 28” and 17”. The overall phase correlation with the glycosidic angle is visible in Figure 5. This feature is also clearly seen in recent decamrr crystal structures ((:rzesko&ak et al., 1991) as well as in the earlier dodecamer structure (Fratini rt al.. 1982).

Apart from the impact of sugar pucker on the backbone dihedrals. we have also searched for all strong correlations involving helicoidal parameters. hackbone parameters and mixt)ures of these two classes. The results bring to light a number of con-rlations that hold independently of base srquenc~ and thus reflect the underlying mechanics of the I)?rjA double helix. Helicoidal parameter uorrrlutions involving the pairs propeller-inclination, twistslide and rise-inclination (wit)h C’o,,-rndo sugars only) are shown in Figure 6. Sate that in the lat’trr case the inter-base-pair parameter rise has been compared with the mean inclination of the two associated base-pairs. Tt is also found that roll and cup are st’ronglg correlated when no O(,,,-endo sugar involved (Fig. 6). Recent crystallographic is decamer structures obtained hy t*hr group of Dickerson show the twist-slide correlat’ion we observe, but also detect relat’ions bet,ween the twist and either roll, rise or cup which. in our case. are limited to dinucleotide junctions involving OC,,,-endo sugars. It is also remarked that correla,tions between 6 and < or between 6 and twist’ found

-I60

-120 Zeta

-80

(deg I

-160

.A Ill/111 !)I! II1 -140 -120 -100 -80 Zeta (3’ we)

(deq ;

Figure 7. (‘orrelations involving thr backbone dihedral c (the corrrlation coeffioient~ is indicated at the top of csac1-r diagram w-ith the number of’ data points used anti the symbols +. s and o are defined as for Fig. 1).

in the dodecamer C(~C(~AATTCIG(‘G (Fratini rf ~1.. 1982) are no longer found by our simula,tions. which also seems to be the case with recent drc~amer crystals (Grzeskowiak ef a,l., 1991). In t>erms of backbone dihedrals. four c*orrelations are seen (Fig. 7): [ with either E or 1 (in the case of X class high amplitude C’(,,, -wxlo sugars) and [ n ith twist or sugar puckering amplitude (in the (me of’ at1 c o,,-~ndo suga,rs). (c)

C’onformatior~a,l

sub-status

As t,he data presented above suggrst, sugar puckers are intimately involved in explaining t)hr existence and certain structural propert,irs of t)hti stable conformations we have obtained. Using t,hr one-1et)ter code we have introduced in sv(*t ion (a). above. w-e caan define dinucleotidr junc~tions 1)~; the two letters corresponding to the suga,rs resl)ectlvrt>found at the 5’ and 3’ side of t#hr junc%ion. Since wt always tind high amplitude C’(,,)-undo (X) or O(,,,-rndo sugars (E) separa,ted by low amplitude C(,,,-endo (8) puckers we observe only four wtcxgories of junction:

sx 9s SE ES

(S : q,,,- rndo + x : c:(*,)-fJndfi) (X : c(,,)-end0 -+ s : (‘(2,,-rnf/o) (S : Co,,-endo (E : (I(,,)-endo

-+ E : (ICI,,-rndo) + s : (‘,~,,-rntlo)

This cllassificat’ion immediately explains ence of the local conformational minima oligomers. since, as Table 4 shows, each tion of a given sequence represents a new tion of junctions. Note that the junctions

thr> existf’or our six conformacombinaof the two

Conformational

Sub-states

Table 6

Table 4 Min,imal

energy conformations classiled in terms of sugar puckers I

2

3

(CQ ) ,4

yxs SXJ

sx X8

SE ES/

(‘I-A),,

ss

xs

ss

sx

SX xs

SE ES

Sequence

4

as sub-states ;i

li

SX ES

W),,

sx xs

xx Sh1

xs SX

SE ES

SE XS

((:A),,

sx ss

ss SE

SS SE

SE XY

XE ss

(AA),, ((:(:)I5

783

in B-D,VL4

Rangen

‘is ss Jh Sh

S. low amplitude Vc2,,- endo: X, high amplitude t ),,,,-mdo: s. intermediate between S and X forms.

(&endo;

E.

strands at a given dinuoleotide level may be identical (e.g. C‘G, SX : SX, writing both junction in the 5’ to 3’ sense) or mixed (e.g. TGz SX : XS or TC, XS : ES). Tt can. however, be noted that only three involve conformations two Co,,-endo sugars attached to the same base-pair (TG,, GA, and GA,). The SE : ES jumtion is clearly forbidden; since this would imply the existence of base-pairs S-S and E-E. the latter of which would have two puckers, whereas such puckers are 0 (,,,-endo observed only for pyrimidines. Special cases occur for the homopolymers, which are forced by the imposed symmetry t’o adopt uniform puckering. These sugars are found to lie bet,ween the S and X : (I(,,,-endo categories. Similar puckers are found for the c~onforrrlat.ion TA 1. If’ we now look at the conformational properties of the junctions. it is found that each has specific features rega,rdless of the base sequeme involved. Table 5 shows that SX. ES and SE junctions all belong to the II, backbone family, whereas XS jumtions t,end towards the II,, form (although we do not observe full transitions to t,his backbone conformation). Note that H,. which is t.he dominant conformation in solution (see, for example. Kaluarachchi pf al.. 19!11). implies E trans and ; gauche . whert,as, in f?,,. E is gauche- and [ trcr.ns (Gupta of a/.. 1980; Fratini et al.. 1982). Both i anti I: have distincat ranges for each junction type

?f helicoidal parameters as (I fiknction of junction type (deg.)

and t.he x values at either end of the junctions are also pre-determined. Sugar amplit,udes are also fixed, since S and E puckers exhibit low amplitudes and X puckers exhibit high amplitudes. As noted previously, the introduction of an (I~,,,-endo sugar fixes the backbone conformation in the same very limited range on both sidesof t,he sugar (i.e. SE and ES junctions are similar). in contrast. $9 and XS junctions have different ranges. For dinucleotide steps where both strands adopt the same junction types, we can go further and predict a certain number of helicoidal parameters as shown in Table 6. These involve twist (relative to the mean value for each dinucleotide sequence) and slide and. in the case of SE and ES junctions, also rise. roll and cup. From Table 6 we can also note that, in terms of twist and slide, the rffect,s of SX and SE junctions go in the same direction and this is true also for the combination of XS and ES junctions (alt.hough ES hardly intiuences twist). The conformational properties of dinucleotide steps nit#h suc*hcombinat.ions can thus also be predict’ed. For other mixed j,unctions t’he conformational properties are intermediate, generally being dominated by the junction that is t,he most clea.rly characterized. Recent high-resolution crystallographic. H-DNA decaamerstructures have been checked in the light of the above findings (PrivC et al.. 1991: Grzeskowiak et a/.. 1991: note that a Lit’h decamer studied in the latter publication has not been considered since it shows important differences in strand conformation despit.e the inversion symmetry of its sequencbe). These c~onformations show good c*orr.elations with our findings, notably for the SX (relative twist. - 12 t.o -6: slide. -0.6 to -92) and SS junctions (relative twist. 2 to 11: slide. 0.2 to 0%: Table 6). Note that slides in the decamers were ~&ulated with respect to their overall mean value which is

Table 5 Krrn.yes of backbone

I I: x (5’. side) x (3. sidr)

dihedr&

and of the glycosidic nnylrs dinucleotide junction type

(deg.)

as n functio~l

of

7x4

.w. l’oncin

unusually posit’ivc (0.9 A). SE occur only t~liret~ tirnes in these decamers and are thus not statis tally significant but t’hey nevertheless show good agrrernent, for slide ( - 0.5 to -WI ) and rise ( -0.3 to -0.1). twist and roll are. however, variable in these cases.Only a single ES junction is present and it is thus not considered. Overall. of t,he 18 unique double strand junctions contained in the three dreamers. 11 can be classed in our categories (although this task is complicated by the absence ot sugar amplitudes) and all these are in good accord with our predictions. It) may be remarked that n.m.r. studies find yualitatire coupling between twists, sugar pucker and phosphate conformations within alternat,ing base sequences (e.g. see Uronenborn et nl.. 1984: Sklenar cf ~1.. 19X9: Schmitz ct al., 1990: Gochin 8r .James. 1990). but t,herr is not, as yet. enough quantitative data t,o attempt detailed correlat’ions with our predictions. It is finally not’ed that earlier t)heoretical studies have also noted a coupling between sugar pucker and twist. 0 (,,,-errdo (E) sugars leading to reduced twist) (Kollman et nl., 1982; Zhurkin Pt nl.. 1990) in line wit’h our SE junct~ions. but t,hesestudies did not distinguish two classesof Co,,-redo puckering. (f) Jhse-pair

stacking

Table 7 lists the calculabed base-pair stacking energies and their four components as defined in Figure 8 and in the order Pyr-Pur. Pm-Pyr, Pm-I’ur. The first’ point) to not,e from t’hesedat,a is

et al

Figure 8. A diagram defining the componentsof’ the stacking energy.

that the same type of stacking for a given dinuclrotide step is often found in several different oligomer conformations. In such cases.the numbers in parentheses indicat,e the range of energies for each component of the stacking and the last’ column lists the number of different, conformations involved. For stackings found in a single conformation, the name of the conformer is given in the last, column. It’ is striking to note that, for the alternating dinucleotide st’epsCpG, GpC, ApT and GpA. iden t,ical stacking is found in all conformations associated but with very different helicoidal parameters. A geometrical investigat’ion of these cases showed that the relative position of the two base-pairs forming the junction are almost identical when the stacking energy and its components are identical. What we set in these casesis therefore a change in backbone conformation that places a fixed st.acking geometry in a different spatial position with respect to the helical axis system and, in this way, gives rise to changed helicoidal parameters. An overview of the stacking component’s shows

Table 7 S’tackin,g

GX ('.(i

energies

-

i.8(

and

~ I I~r)(W) - H5(02)

(:+‘ T.r\

- 1 lM( -10.1 -8.X

14))

(Y: c:x ‘I’,;\ .A.T ‘I’...4 G.(

- I 3q

12)

A.‘1 (X’ 1 :.c ’ .-\.I

~ IO~O(lb4)

Values indicated.

components

(kcallmol)

to the

of stacking

f or each

type

of dinucleotide

step

14)

.\.T T,.-\

G.(’ IX’ A.1 A.‘1

their

- IWl(1~0) - 11~5(M) - 11.1

-!M~((~4) - x.5 - j.7 -9.X - I IfF

in parentheses

correspond

range

for the

number

of conformations

((‘onfmn)

C~onformational

13.51

’

I

GCG CGC ATA

GCG CGC ATA

GTG

TGT

AGA

GAG GGG AAA]

Tt,i

GIG

TGT

AGA

GAG GGG AAAi

CGC ATA

TAT

GIG

TGT

AGA

GAG GGGAAA

GCG CGC ATA

TAT

GTG

TGT

AGA

GAG GGGAAAi

El1

GCG

1

TAT

Su~b-states

Figure 9. Histograms of the groove geometries (K). I’alr shading intlic~atrs thr presence of O,,,)-fJ?/dosugars.

that in Pvr-I’ur (5 to 3’) dinucleotide steps, st’acking is do&inatrd by the interstrand Pur term (2-4). with the exception of TpA, which is stabilized by the two intrastrand component’s, Pur-l’yr steps are dominated b>- int,rastrand stacking l-2. 3-4 (partictulari~ strong in the case of Cpc’) and the oni? st)rong int.rrst,rand term is seen for the T’ur-Pur (l-3) interaction in GpT. Finally, Pur-Pur steps always have a strong intrastrand Pm-Pur component. but are additionally stabilized by the intrastrand Pyr term or the 1-4 intrast.rand term. XpX is unique in having a st,acking interaction almost eyuaii~ dist.ributed bet,wern the four base-base components. Tt should be remarked that earlier theoretical studies (Tilt,on cjt ~1.. 1983), although leading to mucat stronger overall stacking energies. show similar distributions rjt’ stacking components, with the exc*ept,ion of t htl AA st,ep. which shows A dominancat of the l---2 and S-3 terms. Referring again to t,he junction types discussed above. it is found that’ czhanging a bac.kbone juncst)ion does not necessarily change the stacking (e.g. (:pC. C’pC. (;pA and ApT noted above). For the remaining aiternat.ing dinucieotides, changes in stacking are always associated with cahanges in the stronger stacking being purine sugar puckering. linked to (‘c2rI~ rndo conformations. It can also he rernarkrd that within groups of similar stacking. similar helical twists are also observed. It should also be noted that st,ackings involving XS junctions. which t#end towards t)he H,, form. are notab weaker. once again in agreement with experimental findings (( :rzeskoa-iak 4 al.. 1991: (‘ruse Pt nl.. 1986).

in

K-IjAVA

785

(g)

Chove

ge0mfhr.s

The hist’ogram in Figure 9 shows the widths and depths of the minor and major grooves measured at the level of each base-pair with respecatto smooth curves passing t’hrough t,he phosphorus atoms of hhe backhones and. in the case of depth. using a simplified nactangular model for the base-pairs (Stofer & Lavery, unpublished results). The histogram is organized in the same way as that in Figure 3, showing the intra-base-pair parameters. Minor groove Bwidths j;1ave a n4ativel~ limited variation of 11 ,A to 13 A, the only value outside this range (10 A) being obtained with t,he (G(i),, oligomer. whicahhas a high negative base-pair inclination. The (AA), oligomer has a below average width. but is not as low as that seen in the centre of the UKKAATTC(XX dodecamer carvstai structure (Fratini uf crl.. 19%!). Tt can be noted by the shading of thr, histogram t,ha.t the presence of O(,,,-endo sugars generally leads to wider minor grooves. Minor groove depths are* only slightly rariahle. ranging from 3.5 ,A to 5.5 h. Major grooves nre more variable. 5vidt hs varying from I.5 A to 23 A with the largest value for (CC:),, and large values for the seyuences ((l(i),, 11nd[XC:),. Major groove tlepths vary from 4 .A to IO A. the miIjot.it.~ $)f c,onformatioirs newr’l hrirss lie at roughly 7 ;-\. Srvfhral caorrrlations involving groove peometq are found. Incslination affects the widths of both proovrs, positive inclination leading to wider minor proov~esand narrower major grooves. As should be rxpetrted. sdisp directly mfluences groove dept’hs. since this variable corresponds lo displa(aemrnt of the b;lstl-pair along the local ps~~~do-~i-+ asis. Propeller has a sm’ali intiuence on maJor groove width. but does not show any c~orrelation nit,h minor groovr widt,h, although this parameter has ofirn i,een proposed as an explanation oft he na.rr’ow proorl:~associated with XT tracts. Finally. with pure (‘(Z,,-P~~d~jsugars. increasing rise rlrc~rc~asesminor proovc.’ width. while in the prrserrc’e of O,,,,-rndo sugars. major groove width incrc~ases\\,ith more nrgat.ive .rdisp.

4. Conclusions This theoreticsal investigation of the (*onformation of II-I)SA oligomers with repeating base sequences brings to light several results that Irad to a new victw of DSA fine structure. Firstly. each sequence. with the exception of bhe homopolymers. gives rise to sevr>raidistinct st,able conformatmns belonging to thrs H-farnil\-. These conformations. which have sirnilar stabilities. can be characterized as sub-states foih)wmg their sugar puckers. a-hicshc-an belong to two groups wlthin CC,,,-ado or to 0 (l,,-r~~do (in t‘he chaseof pyrimidines). The overall structural properties of the simulated oiigomers and the caorrelations existing between the struct,ural parameters are in good agreement with available experimental data. For any given conformational sub-state. the

sugar puckers are found to fix. to a large cst,twt , thci conformation of thr intervening phosphodiestvr hackbone and a number of the structural charactwist,ics of the corresponding base-pair step (notably. t.wist and slide) regardless of the base seyuenw involved. In contrast. with only a few exceptions, structural parameters do not appear to be fixed dirwtlg by either dinucleotidr or trinucleotide haw sequences. This would suggest that DNA fine st.ruv turr is det,ermined by relatively long-range sequww taffects (at least at the tetranucleot,ide level) that determine the sugar puckers to be adopt’ed. These sugar puckers then, in turn, make a drvisive contrihution in determining the structure of each hww pair step. \Vhet,her B-11X.4 in solution finally represent.s an equilibrium bet,ween different, conformational sub stat,es. as our present energy differences would suggest, or selrcts one dominant form is a question t.hat. cannot he answered at. present. This information will require determination of the energy harriers hetween sub-stat’es and, beyond this. the dynamics of the transitions between sub-statues. its wrll as their equilibrium populations in the prewnw

of solvent and c~ountrrioris:. Its will also lw ntb(*(w4ilr) t.o SW whet.her tracts of (list inct subst.atfLs art’ xt ahlc within irregular hase seqwnws. (‘orrrlations \vit h available experimental data nevrrthrlrss suggest that the sugar pucker c~haractwizc~d c~onformat ions we ha.ve dcsarihed are r&vant to ii tnore clf*iailrtl view of H-I)KA hehaviour. These findings suggest t\\o other points that ma? hr of experimental int.erest. Firstly. it seems likely that’ modrls of I)IVA strnctjrwe based on dinu&wtide sequence effects alonc (which has often hrtw t hr c*ase in dis;c,ussing I)NX hcnding) are unlikvl!to succreetl. ,Src~~rtdly, sugar pucker appears t.o hr crnt,ral in Mining DNA structurt. This is particularly important in r1.rn.r. stuclif5. w.Iifxw t tie present results imply that it, is vfary important to distinguish ac,curately tw1wern pu(skrrs. hot h in terms of phase and amplitude and. notahl~-. to distinguish the two distinct c~lassrs of ( ‘(2,,-~~jdo puckering that. WP de&t. In t,his resfwc*t ii is irnf)fw tant to notr that. because of sug~~r-~)ac~lit)or~f~ dihedral cwrrelat.ions. this distin&on rn>Ly tw achieved irltlirfv~tl!- using “‘1’ data. which fmctblf~ t hr phosphate fmsit,ic-m to I-)fl tlrtrrrnirwtt.

Appendix Full

Axis/lntra:

Structural

Parameters

for the Optimized

(Y: G4’

xdisp - 19 - 13

ydisp WI - 0, I

Inclin - *F.ff - 5.6

( ‘pG Gp(’

Shift 04 0.0

SlidP ~ 02 0.2

Rise 3.5 3.5

(‘pG Gp(’

(Ihi -120 - 116

Epsil - Ii3 - 173

Zeta -110 -,“i

Phase I64 157

Ampli 3i 44

t’ucker (’ ,,,,-fwlo ( ‘,z‘,-“‘d”

Ikpt h 4.4 44

(Major)

\Vitlth 1n.a I 9,.-i

xdisp - 15 - 1.6

ydisp - 0.3 0.3

Iwlin -0-9 - 0.9

Tip w:! - 0.2

Shift 04 (I4

Slide 0.6 -@6

Rise 3.4 3.4

Tilt 04 0.0

(!pG GpC

(‘hi -113 -111

Epsil -166 -Iii

Zeta - 133 - 104

Alpha - 65 -67

(’ c:

Phase 1% Ii8

Ampli 44

Intw

sb ugar: (’

(;roo\‘cs.

(Minor) (‘4: (X’

Tilt 04) 03)

m:, Axis,‘lntra~

cx; (X‘

Int~er.

Uackhonr:

Sll&W:

38

Pucker Cc,,,-rndo (:,,,,-end

Oligomers

Conformational

Appendix Width 11.4 11.4 (‘(i,

Depth 4.6 4.6 (WY

Axis/Int,ra:

(continued)

(Major)

Width 17.3 17.2

xdisp -2.3 - 2.3

ydisp -04 04

In&n 1.9 l-9

Tip -0.4 W4

(‘p( : Gp(’

Shift 0.0 04

Slide 0.9 - 0.9

Rise 3.1 3.5

(‘hi -143 - I10

Epsil ~ 172 - 173

zeta ~ 96 - 100

Phase 87 173

Ampli 35 37

Width 13.0 13.0

Depth 4.2 42

ISackhone:

sugw (’ (:

TA,

Depth 65 6.3 (Energy

X:GC’GCG(:GU:

( ‘4 : (l4’

Intel

787

Sub-states in K-DXA

= -8160)

Buckle - 6.4 64

PlWpd - 4.0 -4.0

Tilt 04 0.0

Roll 0.9 -0.9

Twist 3@4 :3u2

Alpha -64 -72

Beta -179 173

(:amma 59 34

FVidth l&l 181

Depth 7.;

Pucker O,,,,-mdo

(‘(2,)-Pndo (Major)

7.6

(Energy

TATXTATATATATA

= -562.1)

xdisp -3.6 - 3.6

ydisp 0.0 0.0

Inclin 12.0 12.0

Tip -2.9 “9

Buckle 3.0 - 3.0

Propel -4.1 -4-l

Shift 0.0 00

Slidr 0.0 00

Rise 3.0 2.9

Tilt 0.0 04

Roll 54 -58

Twist 339 33.4

,+p’r

Chi -113 -113

Epsil -172 -I72

zeta - 106 -113

Alpha -73 -65

Beta -175 177

Gamma 50 56

T A

Phase 152 153

.~rnpli 40 39

Pucker (‘,,,,-end0 (‘o,,-endo

Width 12.6 12.6

Depth 36 3.6

(Major)

Width 15.6 15.6

Depth 10.2 10.3

Axisilntra:

Tp.4

Sugar-

7‘. ‘ 4 A.‘1

winor)

TATATATATATATA

TA,

(Energq-

T.A A.1

xdisp -23 -2.8

ydisp @l -0-l

In&n 6.”d 6.2

Tip - 3.9 3.9

Tp.l Apl

Shift 0.0 0.0

Slide - 03 0.3

Rise 3.3 3.0

ISackt,one:

Chi -112 -116

Epsil -173 -I71

zeta - 103 -121

Sllpar:

Phase 160 151

Ampli 38 41

Width 121 12.1

Depth 4.1 4.0

Axis/Intra:

Inter:

Grooves:

(Minor) T.A A.‘1

Axis/Intra T.A A.‘1

Buckle 4.4 -44

Propel -3.9 - 3.9

Tilt, 00 0.0

Roll 7.7 - 7.7

Twist 32.6 36.2

Alpha -72 -62

Beta -1i7 174

Gamma *il SH

Width 17.1 17.0

Depth 8.7 8.8

Pucker Cc,,,-do

(‘ (28,&O (Major)

(Energy

TATATATATATATA

T.4, xdisp -2.0 -2.0

.vdisp - 0.3 0.3

= - .5597)

Inclin 3.2 3.2

Tip - 1.0 1.0

Buckle 1.3 - 1.3

= - 556.3) Propel - 2.7 - 2.5

Appendix

((‘o~L~~II((,(‘~I)

Shllf (I.0 t I3 I

( ‘hi I I3 -ll'L

_

I'lIilS~ t*;I 177

‘I’(:‘I’(:‘r(;‘r(:T(:Tt

;I?:

.rdisp - 1.4 - 1.3

ydi sp

Shift 0.1 -WI

Slide

K isr

(Hi --(I4

3.2 3.2

lkpth

(Ma.jor)

._ 0.2 0~3

I ticlin 5, I 44

( ‘hi - IOH - IOH - 1 Ofi --II:!

I4’idt.h I I.8 I I .!f

4.8 4.5

T(:T(:T(:T(:T(:‘I’(:TCl xdisp -2.0 - I.0 Shift, 1.1 -1.1

Slide 0.2 - 05.2

Rise 34 3.0

Conformational

Sub-states ,in B-DNA

Appendix Backhone: Tp(i (:p’l Ap( ( (‘pA

Chi -112 -114 -112 -110

Epsil -171 -169 -172 -162

T (:

Phase 161 147

Ampli 34 4%

Width 11.3 122

Depth 5.7 3.7

sugar:

(,Minor) T.A (X‘

TG )

Zeta -97 -131 -94 - 14% Pucker C,,,,-end0

Ccz,,-endo (Major)

Alpha -67 -61 -66 -65

Beta 172 171 171 174

Gamma 5i 5x 56 54

A (’

Phase 184 146

Ampli 34 45

Width 163 15%

rdisp - PO -1.9

ydisp 0.0 -0.2

Inclin 0.1

Tip -1.6 -0.1

Tp(i (:p’l’

Shift 0.0 0.0

Slide -W% oc2

Rise 33 34

Tilt - 2. 6

Tp(i Gpl Ap( ( (‘I’.~

Chi -113 -118 -110 -119

Epsil -174 -172 -171 -172

Zeta - 107 -120 -125 - 108

Alpha -67 -61 -64 -68

vr t:

Phase 163 152

Limpli 3-i 4%

Pucker (‘,,,,-P?lh (‘,,,,-UtdO

.A (‘

Width 11.3 11.3

Depth 45 44

(Major)

(Minor) T.A (a( I

T( &,

-26

26

Width 184 1 x.0

TGTGTGTGTGTGTG

Buckle - 12 -0%

684%) Propel - .%!I 0.0

Roll 1.4 -1.4

Twist 33.3 38~2

Beta 178 177 174 -177

Gamma 55 .ii 59 54

Phase 160 165

Pucker (’ ,,,,-endo (’ ,,,,-mdo

Depth 7.0 7.2

(Energy

691.5)

xdisp -1.8 - 1.6

@iv

T.&J (X‘

- 0.4 0.5

Inclin 65 38

Tip -1.0 1.2

Buckle -2.2 7.8

Proprl -17.5 - 12.9

Tp(: Up’1

Shift 0.2 -0.2

Slide 0.9 -0.9

Rise 31 3.3

Tilt -2.7 2.7

Roll 2.2 -2.2

Twist 390 30.9

Tp( ; Gpl Ap( ’ (‘p.4

Chi -134 - 106 -104 -142

Hpsil -171 -172 -173 -174

Zeta -93 -104 -97 -95

Beta -180 169 169 -179

Gamma 58 60 -59 60

1 G

Phase 91 174

lmpli 32 35

Pucker O,,;,-endo (‘,*,,-WZdO

A (’

Phase 178 82

Ampli 36 37

Width 13.0 13.2

Depth 4.6 4.3

(Major)

Width 161 157

Depth 7.2 7.3

Axis/Intra

Inter:

Backbone:

Sllgar:

(Minor) T.A (X’

TGTGTGTGTGTGTG

TG, Axis/Intra

Alpha -65 -69 -70 -63

(Energy

= -6951)

T.A (a(‘

sdisp -1.5 -1.3

ydisp 0.0 0.7

Inclin 45 1.5

Tip 21 41

Huckle - 1.9 1.6

Propel -138 - 12.2

TpG GpT

Shift 0.2 -0.2

Slide 0.7 -0.7

Rise 31 33

Tilt - 3.0 3.0

Roll 2.0 -2.0

Twist 41.9 30.8

Inter:

Pucker (‘(3,,-exe (‘,,,,-mndo

Depth 56 7.1

(Enerp~

T.‘-\ (H Inter:

SuptLr:

(continued)

TGTGT(:TGTGTGTC:

AxisiIntra

789

Pucker

(‘,,,,-endo O(,,,pdO

Appendix Epsil - 165 -. 17li -17% -I75

‘I)‘(; (:pT A@’ I $‘A

zeta 134 I0.i -- !46 -~ 9-l

Phase 156 IX1

‘1 1: T,X

(contirwd)

(Minor)

(X’

LVitlth I 64 1%

T(:T(‘T(“r(:TGT(:T(: r T

TpG (:pT

rdisp -1.6 - 1.6

ydisp -lb7 0. I

Shift 0.0 0.0

Slide 0.7 - 0.i

I ‘hi - 133 -IO6 - 105 -114

lCpsi1 -I73 -171 -177 - 16.5

lnclin 6.3 -5.3

(Minor)

Tip -. 2.0 - 20

Width 12.7 12%

(ix A.7

xdisp - 1.5 - 1.7

.!disp 0.3 -02

Shift 0.2 - 0.2

I~ucklr 1+i 6.2

Tilt - 1 .o I.0 Alpha -64 -68 -67 -67

.\ (’

T.A I:,(’

Drpth 6.7 Ii.7

(lhW#,\

Phase 91 175

T t:

.Zmpli 3-l 3x

A (’

b’idth 12.0 12.4

TG,

.\I]h -- Ii!4 ~- Ii6 -6X -lil

Twist 41.5 30.7 I3eta ~ I79 I 65 1x0 174 Phaw IX2 I50

\Vidth 15-t IS.5

Depth 7.0 7.1

Inclin -co -4.5

Tip (k.4 03

Buckle - 50 -5.x

Slide 0.5 - 0.5

Rise 34 3.5

Tilt - 1% I.6

Itoll 02 -0.2

(‘hi -119 ~ 110 -120 -116

Epsil - 1 io -175 -174 -171

%&I -128 ~ I03 - 108 -125

Alph” -63 - 63 - 65 - 63

Beta I79 176 -178 17”

Phasr I48 I76

Ampli 45 35

Puckrr (1 ,,,,-mdo (‘ ,,,,-rndo

I’ 7

Phase 166 155

Width 107 lo.7

Depth 4.5 4.x

(Major)

M’ A.T

Width 18.7 I x44

Depth Ii.4 6.2

zdisp -2.2 -2.8

ydisp

M‘ A.T

- 04 - 0.3

In&n -54 -2.4

Tip - 0.2 - 1.8

Buckle -5.7 I.7

GpA ApG

Shift 0.6 -0.6

Slide -0.1 0.1

Rise 3.6 3.2

Tilt - 3..i 3.5

Roll 1.5 ~ I.5

UpA A$ VpT TpC’

(: A (Minor)

(iamma -57 61 .i4 34

Ampli 37 44

Twist 34.0 32.3

Conformational

Sub-states

Appendix

SUgW’: t: .A (?&nor) (:.(’ A.7

Chi -119 -115 - 150 -118

IXpsil -172 -1i4 -174 - 170

Phase 151 163

Ampli 43 Ji

Width I I-6 11-1

Dept,h 3.8 4.9

(:A,

in,

B-DN.4

791

(contimed)

Beta

Zeta -120 - 109 -94 -96

Alpha -63 - 63 -61 -68

-175 175 -179 168

Puckel (‘,2,,-enddo (‘ (z,)-P1uIo

(’ ‘r

Phase 92 155

L%‘idth 20.1 mri

Depth 7.6 4.0

(Major)

GAGAGXAGAGAGA

(Energ!

Gammn 32 ix Xi 63 Arnpli 39 37

= -676.1)

GX A.7

xdisp - 1.8 -1.8

ydisp -0.i -0.1

Inclin -44 0.9

Tip - 3.3 - 3.6

Buckle -9.1 -3.9

Propel - 5.9 - 8.7

GpA Ap(i

Shift 04 0.0

Slide -0.6 0.6

Rise 3.5 3.2

Tilt -4.9 4.9

Roll 0.2 -0.2

Twist 31.X x+1

Chi -11.5 -108 -148 -116

Epsil - 17.i - 165 -174 -1il

Zeta - 103 -137 - 90 -98

.Upha -69 -66 - 59 -ti7

Beta -174 170 Ii5 I68

Gamma -50 60 60 61

Phase 176 lR8

Ampii 3x 44

(’ T

Phaw 86 137

\%Ydth 11-i 11.5

Depth $5 4.6

\I’idth 18.1 18. I

Depth 7.0 64

Inter:

Sugar. (: A

(‘.f’ d.7

wnor)

Pucker 0, I ,,-rndo (’ (2,,-rn,do

Xmpli 39 35

Pucker 0 t,,,-~ndo f’ ,,,,-do

675.8)

M -4.7

xdisp - 2.0 -2.1

ydisp 0.0 -0i

Inclin - 0.2 - 0.4

Tip -2.8 - 2.0

Buckle -0.8 - X.6

Propel - 2.7 - 7.0

(:pA Ap( :

Shift 0.1 -01

Slide 0.7 - 0.7

Rise 3.2 3.5

Tilt oc2 -03

ROll - 0.8 0.8

Twist 395 29.2

GpX Ap( : CpT Tp( ’

C’hi -117 -108 -118 -142

Epsil -167 - 175 -170 -173

Zeta - 132 -103 -101 -91

Qha -65 -66 -68 - 63

Beta 177 -180 170 176

Gamma 58 52 60 60

(: A

Phase 150 179

Ampli 44 36

Pucker c(*,)-PnLlo

(’ T

Phase 157 92

AUlpli 34 36

Width 11-8 11-9

Depth 4.4 4.5

(Major)

AxislIntra

Inter:

I%ackborw

Sugar:

(Minor) G.(! A.7

GA,

C’,,,,pdo

Width 18.6 18.6

GAGAGAGAGAGAGA sdisp -2.5 - 1.6

ydisp -0.5 -0~5

Inclin 40 7.8

Tip -4.8 - 3.0

GpA ApG

Shift -1.0 1.0

Slidr 0.0 0.0

Rise 3.0 3.4

Tilt -3.8 3.8

Inter:

7.3

(Energy

G.(‘ A.T

Axis/Intra-

Depth 7.1

Buckle -56 -16.0 R,oll - 1.8 1.8

= -8670.6) Propel - 1.X -123 Twist 33.5 36.1

Pucker (’ ,,,,-crulo O,,,,-mdo

792 -

__--..-

;IJ. t’oncirr

Appendix

et al.

_.-

(continued)

(+I ApG (‘pT Tp( (

Width 122 13.1

Pucker ( ~(3,,-Px0 (‘,,,,-rndo

Phase 1X A2

(Major)

Depth 7.7 7%

(X 1

(Energ,)

(a(

Buckle 3.3

AxisjIntra:

Shift 04

Inter: c:pc: J%ackbone:

SlJgtLr: (4 (:roovrs:

(Minor) M

Roll 0.0

(‘hi -123 -127

Epsil - I75 -174

Phase 154

&npli 42

Zeta -114 -117 Pucker (’ o,,-Pndo

Phase 1.x

\Vidth 104

(F:nerg,v

A.‘,

rdisp -1.3

ydisp 0%

Inclin 63

APA

Shift, 04)

Slide 04

Rise 3 I

APA TPT

(‘hi -98 -113

Epsil -171

-117

Inter:

Backbone,

(:rooves:

(Minor) A.T

Key

to dihedral

)’ 6 E i x

Gamma Delta Epsilon Zeta Chi (Pyr) Chi (Pur)

Buckle I49

zeta

-171

Width Il.3

Pucker (’ ,*,,-WLd(J

= - .(X)3. I ) Propel -184

(iamma

57 .59

-111

.-2mpli 38

Pucker (’ ,a,-mdo

Depth 47

(Major)

Depth 6%

angles:

The authors thank the Association Cancer Research (St. Andrews University. generous support of this work.

for

International UK) for their

References Arnott,

Ampli 42

Depth 3 I

AA, AxislIntra:

Propel I 1.3

S. & Hukins, D. W. L. (1973). structure of B-DPU’A and implication

Refinement for the

of the analysis

of X-ray diffraction data from fibers of biopolymers. .I. Mol. Biol. 81, 93-105. Arnott, S.. Chandrasekaran, R.. Birdsall, U. L., Leslie. ,4. Cr. W. & Ratliff, R. L. (1980). Left handed helices. Nature (London), 283, 743.-745. (‘alladine. C. R. (1982). Mechanics of sequence dependent stacking of bases in B-DKA. J. Mol. Rid. 161.

343-352. Cruse,

W.

B. T..

Salisbury.

S. A.. Brown,

T.,

Costick,

R..

Con,formational

Sub-states

Eckstein, F. bt Kennard. 0. (1986). Chiral phosphothionate analogues of B-DXA. The crystal structure of IPl’((:pS(‘J)(:pSCpG1,8(‘). J. Mol. Riol. 192. 891 905. Dickerson. R. E.. Bansal. M., (‘alladine. (1. R.. Diekmann. S.. Hunter. W. S.. Kennard. 0.. Lavrry. R,.. h-elson. H. (‘. ,\I.. Olson. M’. K.. Saengrr, W., Shakked. Z.. Sklenar. H.. Soumpasis. D. M., Tung. (I.-S.. van Kitzing, E.. \\‘ang. A. H.-J. & Zhurkin. \-. B. (1989). Drfinitions and nomenc>lature of nucleic acid structure parametrrs. .J. No/. Riol. 205. 787-791. Fratini. A. \‘.. Kol)ka. M. I,.. Drew, H. It. & I)irkrraon II. F:. (I!%?). R(Lvrrsihlr bt>nding and helix g[romcitry in a H-l);\ji\ dodecamer: ( ‘G(‘C:A1ATT”‘(‘(X ‘ct. .J. Biol. t’hrm. 257. I-CBXB- 1470;. Fritsch. \-. & IVc~sthof. E. (1990). Minimization and molec,ular dynamics of Z-DIL’A modified by acetylaminotluorrnr. 111 Modelling of Molecular Str?cctures an/l Proprrtic,s (Rivail J. L.. pd.). pp. 627-634. Elsrvier. :imstrrtlam. (:oc:hin. .\I. & ,Jamc~ T. I,. (1990). Solution structure via restrainrd molecular studies of d(A(‘);tl(GT), dynamic.s sinlulations with MIR constraints derived from t>wo-ditnrnsional SOE and doublr quantum filtered (‘OSY exprr,imrnts. Biochrmixtry. 29. llli’--11180 (:ronenbor11. A >I.. (‘lore (:. 11. & Kimher. 1s. .J. (1984). An investigation into thr solution structure of two srlf-c~oml)lrmrntarS DX.4 oligomers. .i’-ti(CGT~~CG) alltl T,‘-tl(A(‘(:(‘(:(‘C:T). by means of KOE measuremrnts. l~io~h~n~. ,J. 221. 723.-736. (irzrskowiak. K.. Yanagi. K.. I’rivB, (:. ($. & 1)ickerson. E. (1991). The structure of H-helical R (‘(;AT(‘(:Al’(Y: anti comparison with (‘(‘h,\(‘(:TT(:(:. .f. IZiol. C’hem. 266. 8861. xxx3. (iupta. (i.. Bansal, M. 6 Sasisekharan. V. (1980). (‘onformational flrxibilitv of DXA: Polymorphism and tiandrtlnc~ss. Proc. :i:ot. Acctd. Sri.. l’.L?..-I. 77. 6468--6490.

Hao. M-H. & Olson; W. K. (1989). Molecular modeling and energy refinement of supercoiled Dh’A. J. Biontol. Strlrrf. JIynam. 7. M-692. Hartmann. 1% Ma!fov, 1%. & Lavcbry. R. (1989). Theorrtical l,redic;ion of base sequenc.r effects in DI$;A. lQq)rrirnental reartivity of Z-DS.-\ and B-Z transition rnthalpies. J. Nol. Biol. 207, 433-444. Hingerty. I for irregular nucleic acids. .I, Hiomol. Struct. J)ynnwr 6. 63, 91. Lal-rry. R. B Sklrnar. H. (1989). IMininy thv struc*ture of irregular nuc4ric acids: vonventions &ci principles. .J. IIiomol.

Strrcct.

Dynam.

6. 655-667.

I,avc~ry. R.. Zakr\vewska, K. & Pullmall. .I. (1981). ( )pt imizrd mc~nopole expansions for t hr rrpresrntation of thv elec:t,rostatic properties of the nucalt,ic ;lcitls. .J. ~‘omput. (‘hem. 5. 363-373 Lavt’l~~. R.. Sklenar. H., Zakrzewska. K. ct Pullman. R. (19%rr). The tlrxibility of the nuc+ic~ acids. (II) the calculation of intrrnal enrrgy and wj,plic*atiorrs to trior~cinuclroticl~~ reprat I)FiA. ,I. f~iwol. strwi. /),yMnl. 3. YX!)~~lOl1. 1~avc~ry. R.. Parker. I. 8r Krndrick. .I. (I986b). .I genrral approach to the optimization of thta conformation of ring molec~ulr~ with an application to valinom!-tin. ,1. f~ion~ol. Strurl. Dynnm. 4. 443 -461. Mrtzlrr. \2’. .I.. \\‘ang. C.. Kitchen. I). I