Structure of a B-DNA decamer with a central T-A step: C-G-A-T-T-A-A-T-C-G

I. Mol. Biol. (1992) 225, 379-395

Structure of a B-DNA Decamer with a Central T-A Step: C-G-A-T-T-A-A-T-C-G Jordi R. Quintana, Kazimierz Grzeskowiak, Kazunori Yanagit and Richard E. Dickerson Molecular Biology Institute Department of Chemistry and Biochemistry, and Institute of Geophysics and Planetary Physics University of California at Los Angeles Los Angeles CA 90024, TJ.8.A. (Received 20 September 1991; accepted 20 December 1991) The X-ray crystal structure analysis of the decamer C-G-A-T-T-A-A-T-C-G has been carried out to a resolution of l-5 A. The crystals are space group P2,2,2,, cell dimensions a = 38.60 A, b = 3910 8, c = 33.07 A. The structure was solved by molecular replacement and refined with X-PLOR and NUCLSQ. The final tl factor for a model with 404 DNA atoms, 108 water molecules and one magnesium hexahydrate cation is 157o/o. The double helix is essentially isostructural with C-G-A-T-C-G-A-T-C-G, with closely similar local helix parameters. The structure of the T-T-A-A center differs from that found in C-G-C-G-T-T-AA-C-G-C-G in that the minor groove in our decamer is wide at the central T-A step rather than narrow, and the twist angle of the T-A step is small (31.1”) rather than large. Whereas the tetrad model provides a convenient framework for discussing local DNA helix structure, it cannot be the entire story. The articulated helix model of DNA structure proposes that certain sequence regions of DNA show preferential twisting or bending properties, whereas other regions are less capable of deformation, in a manner that may be useful in sequence recognition by drugs and protein. Further crystal structure analyses should help to delineate the precise nature of sequence-dependent articulation in the DNA double helix.

KeIywords: B-DNA;

structure;

1. Introduction Synthetic DNA decamers have proven to be much better candidates for crystallographic examination of sequence/structure relationships in DNA than have other length of oligomer, because the singleturn decamers stack in a crystal lattice in a manner that simulates a continuous helix (PrivB et al., 1987, 1991; Heinemann & Alings, 1989, 1991; Yanagi et ul., 1991; Grzeskowiak et al., 1991). Possibly as a consequence, they tend t,o exhibit markedly better order and higher resolution, 1.3 to 1.6 A, as compared with 1.9 to 2.6 A for dodecamers (1 A = O-1 nm). The DNA double helix is both deformed, and made deformable, by its local base sequence. That is, some regions of DNA may be given a particular local conformation by sequence, whereas other t Present address: Sumitomo Chemical (I>ompany, Ltd. Tsukuba Research I,aboratory, 6 Kitahara, Tsukuba. Tbaraki.

300-32.

Japan.

X-ray

crystallography;

T-A step

regions may be made especially susceptible to a change in conformation when the helix interacts with neighbors: a protein molecule, a drug molecule, or an adjacent DNA helix in a crystal lattice. DNA has been shown to be a molecule with a high degree of flexibility, whose structure depends on interactions with other molecules (Travers, 1989). How far can one go in learning the rules for DNA structure from an examination of oligonucleotide crystals? There was a time when attention was focused on the individual step from one base-pair to another, and attempts were made to establish standard helix parameters, such as twist, for each of the ten possible base steps. This proved to be a fruitless endeavor. When one asks what is the standard twist angle for a C-A step, the only proper answer is: It doesn’t have one. A step such as C-A can be either high twist or low twist, depending on the environment. in which it occurs. To the extent that this environment consists of the base-pairs immediately preceding and following, one is driven to the “tetrad

379

~________.__ hypothesis”. th concept that the proper unit 01’ study in I)IVA sec~uenc~:/st’ruc.turr relat~ionships is I hll step in question plus those tha,t lead ad f’ollow. or il stretch of four successivr kmsr-pairs. Tanagi et u/. (1991) established that. there exist 136 uniquch t)ase t.etrads. analogous to t,he ten diff‘rrrnt t\vc) hast:-pair steps. Synthet,ic I>,I;A decamers vary widely. and inexplicably. in the easr with which they can be crystallized. The first t WI, (‘-(‘-A-A-(:-A-‘r-‘r-(;-(: (the GA decAamc>r) and (‘-(‘-A-A-(I-(:-T-T-(:-(: (the (Y: dc~c~amer) were isomorphous. in monoclinic space group (‘2 with fivcl base-pairs per asymmetric unit. (l’riv6 Pf nl.. 1987. 1991). Attempts to crystallize sc~lf-c,ornplcrnc~rit,~~r~ analogs with t,he other t.hrt>cJ \Vat,son -(!ric*k pairing possiMit,ics in thr two c~rntjral pairs: (: ( I. A ‘I’ and T. A, wt-‘rt’ urisat,isfacatory. ‘I’. A gave tiny t,rigonal crystals that diffrac~lrd only to about 7 4: the others gave no cbrystals at all. ((‘. I and I . A tnorp r~crntly have bern su(acQessful.) On thfa ot.hcr hand. what would sertn to IW a morc~ drilstit, alt.erat.ion in sequence. (‘-(‘-A-(:-(:-(‘-(‘~‘I’(Xi. led to excellent csrystals in the same monoclinic* space group (‘2. and to a structurr analysis that caxbended to I.6 .A resolution (Heinemann & Alings. I!##). For reasons that are a mystery. a crystal packing that) is favorable for (‘-(‘-d-A-(I-C:-T-T-(;-(: and (‘-(‘-A-(:-G-(I-(~‘-T-(:-(: is very difIic:ultj for t h(l intermediate srquencr (‘-(1-A-A-(:-(1-T~‘r~(;-(:. It ~ultl be t,hat. only certain tetrad srquemrs art allowed t,o crystallisc within t’hr c*ommon frarnework of (l-(‘~A-x-x-x-x-T-C:-C: because onlv their st!ruc:turrs can adapt t,o t’he specific constraints of that tryst al packing mode. A major alteration in sequence to yield t,he soc*alled KK drcamrr. (:-(:-4-T-(‘-(;-A-T-(1-(:. led to a new crystal packing scheme: orthorhomhic spac’ct group 1’%,2121 with ten base-pairs per asyrnrnet ric unit. again with flntl-for-end helix st)ac+king and a high degrrr of crystalline order ((:rzeskowiak PI nl.. 1901). Decamers of sequence type: (‘-(LA-x-x-x-x‘I’-( I-G tend to adopt t,his orthorhombic, crystal form and will he termed the KK family> whereas those of thf> type (:-(‘-~4-s-x-x-x-T-(:-(: use monoclinic: packing and will hr called the CX family after the Priv6 it nl. representative. The KK family seems to t)r more tolerant of small alterations in the center of thca base sequence than is the CG family. Wr have succeeded in replacing the pyrimidine-purine step at. t,he center of the KK helix wit,h the ot’her selfcomplementary pyrimidine-purinc stq), suhstituting -T-A- for -(‘-(i-, to yield what, will be designated for brevity the TA helix, (X-A-T-T-A-A-T(X. This paper describes the results of a crystal structure analysis of the TA helix at a resolution of 1.5 A.

2. Methods Crystals of (1.G-A-T-T-ALA-T-(“-(: solution containing W 14 M-magnesium (pH 69) and loo/,

were grown from a

0.34 mM-DNA decamer double helix. acetate, O-34 mw-spermine.4HCl (v/v) 2,4-methylperrtanediol. by sitting

Structure of a IS-DNA Decamer: (‘-C-A-T-T-.-1-A-T-C’-(:

(b)

Figure 1. TA IWSUS KK difference maps in the vicinity of T.5. A16 or C5. G16 respectively, showing positive and negative peaks at, thymine methyl and guaninr X-2 amine sites. Origin at upper left., s downward from 0 to l/2, y to the right from 0 to l/2. Superposition of 3 sections at 8. 7 and S/52 in z. Difference density contoured at intervals of WO e-/A3 from --IQ0 to +19Oe-/A3. zero contour omitted. Positive contours continuous; negative contours broken. (a) TA data used with KK model, Fourier coefi(Gents: (Fz* - FrK)exp(idKK). Sate the positive peak where thymine methyl should be, and a negative peak where guanine S-2 amine is not indicated by the TL4 intensity data. The la level is @111 e-/a3. (1)) KK data Fourier coefficients: used with TA model, (FrK- FTA)rxp(i&‘rA). Note the negative peak at rrnwant,rd thymine methyl. and positive peak at missing

3X1

the number of FO terms per solrrrlt rnolt~~ulr found (including magnesium complexes) is virtua.11~. tdentiral for both structures: 339 experimental rnrasurrmrnts per solvent peak for TA and 32.7 for KK. TN-rlvr of the solvent peaks occ~upy the same position within 0.25 :\ in these st,ructurrs; another 16 lie within 0.3 -4. anot,her 23 within 0.75 LAand still another 13 n-ithin I.0 .\. The best match between solvent molecules in thte II strucbturen is found with the magnesium complex. water ~t~otecules in th(a minor groove. and the water tnolec~uh~sthat bridge different hrlices in the crpst.al. Water I~IOIP~~IIIW wit,hin the major groove csorrespond less well. The similaritv in X-ray survey pre(*ession photographs and unit ~rll dimensions between KK and T.2 clec~amrrs ted us from the outset, to expect few tlifferrrtc~rs in c*rystal struc+turr. Atter exponential scaling of t,lity P1L’3 POterms above z’a that arr common to both data stst::. the residual dif%rrn~r. Rdata= EIF, -F21/21CF,. is only 3).X”,,. Of 104 I)?;:2 atoms in racah of the 2 drc.anrers. I ‘I’;\ itntl K K structures. each of tht> final (ho-ordinate sets was refined against thr S-ray data of’thch other helix. (!Jm- PC) diffen~nc~e maps (Fig. 1) wtir(l c*al(.nlated and examined to see whether the differences between .+I.T and G (’ base-pairs. thymine methyl in the major groove and guanine amine in t,he minor groovth. would be discernible. TCJavoid obsc.urinp the differenrr maps with Lvater peaks, th(> ion and solvent positions were inc*luded with I)SA ttw moat atoms in the F c~al(~ulations. In both c*asw J)r.ominent positEve and negative differenc,tA peaks in the differrnee tnaps were those that would br t.xprc%etl in terms of addition or removal of thyminra (‘ .> rncxthyl and guaniml N-r’ amine groups. The asymmetric unit of the final refined model contains 1 double-helical dreamer of 404 1)X.4 atoms. 108 water molecules (trrat,ed as oxygen atoms) and 1 hexahydrated magnesium cation. Mg(H,O)z’. The original F, S-ray intensity data and the final refined c.o-ordinates hare been de1jositt.d with the Rrookhaven Protein Data Rank. and art’ c*urrently available upon request (ent.rirs R 11)4!#F and 11)49. respectively).

guanine amine. The la level is 0.126 em;B3. These positive and negative peaks are the largest residual feat,ures on either of t.he difference maps. The trio of bonds radiating from a cnommon point in the minor groove below marks the hydrated magnesium cation. whose mrt,al center lies on adjacent se&on 5. Note the complete absence of differenc*r contours around the site of the well-fitted phosphate group at the lower right.

3X2

.J. I?. Quintana

et al

Figure 2. Stereo CPK drawing of the structure of the TA helix (C-(:-,1-T-T-A-,~-T-(‘-(:), groove at t,he cent,er of the helix. The groove narrows appreciably toward each end. 3. Results parameters were calculated the SEWHEL91 program the last author at RED@UCLAlJE.

Local

helix from

helix

using

O-4’ O-4’ P P

for the TA (available or from

I 2 3 4 5 6 7 8 9 IO I 2 3 13 12 II 20 IQ 18 17 16 15 14 13 12 II 2 3 4 5 6 7 8 9 IO -- 2 3 13 12 -- 20 19 18 17 16 I5 14 I3 12

Figure 3. Minor groove widths in the TA helix (filled circles. heavy lines) and KK helix (open circles, light lines). as measured by shortest P-P and 0-4’-O-4’ distances across t,he minor groove. Phosphorus and sugar numbers are given along the horizontal axis. The center of t,hr helix is at 0-4’-O-4’ vector 7/17. and the junctions h&ween stacked helices are at O-4/-0-4’ vector Z/12, both king regions of maximum minor groove width (tall arrows). Minimum groove widths occur halfway between. at P-P vectors 5/2O and lo/15 (short arrows). The length of 1 helix is given by the distance between the 2 outermost long arrows.

viewed

ink

the wide minor

the Brookhaven Protein Data Bank). These values are listed in Appendix Tables Al to A3 in a format identical with that used earlier for the monoc~linic decamers (Prive et (LI., 1991) and the KK helix (Grzeskowiak rt al.. 1991). Also tabulated for each parameter are means and standard deviations (AV and SD). and mean symmetry differences (1~1) between the two sequentially identical but c~rystallographically distinct ends of the helix. Non-zero values of this latter quantity cannot be attribubed t,o base sequence effects, and can result only from some combination of c>rystal packing forces and/or experimental uncertainties in refinement (Dickerson et al., 1991). For comparison purposes, the quantities AV, SD and I>1 for the KK decamer are repeated from Grzeskowiak rt nl. (1991). In general. mean symmetry differences IIT are similar for both helices, usually slightly smaller for the KK helix than for the TA, as expected from the greater number of X-ray reflections per basepair: 511 for the KK helix VRTSUS379 for the TA. Because the mean symmetry differences DI are not uniformly zero, the issue of crystal packing bechomes significant. (a) Structure

and crystal

packing

in the I’A

decamer

Crystals of the TA helix are essentially isomorphous with those of KK. Figure 2 shows a view of one TA decamer, facing into t.he minor groove at thr center of the helix. As with KK, the minor groove of the TA decamer widens in the center (central long arrow in Fig. 3), rather than adopting a narrow minor groove geometry with a spine of hydration as observed previously in the A-T-rich dodeoamrrs

Structure

of a B-DNA

Decamer:

(‘-C:-A-T-T-~4.,4-T-C-G

383

(b)

Figure 4. Stereo drawings of 4 columns of stacked TA decamer helices, viewed along the crystallographicb axis, with the a axis from left to right and the c axis from top to bottom. (a) Skeletal representation. This is a more extended view, analogous to Fig. 2 of Grzeskowiak et al. (1991) for the KK helix. (b) Space-filling representation. This drawing illustrates how the minor grooves join to build continuous diagonal channels through the crystal.

(Nelson et al., 1987; Yoon et al., 1988). Again as with KK, the TA decamers st.ack atop one another along the c axis of the crystal to form pseudo-continuous helices arranged in a square array, with extensive lateral contacts along the a and b axes of the crystal (Fig. 4; Compare Figs I to 3 of Grzeskowiak et al., 1991). The space-filling stereo drawing in Figure 4(b) illustrates an interesting feature of this mode of crystal packing: minor grooves of neighboring helices form continuous diagonal channels through the crystal. Table 1 presents close contacts between DNA molecules in neighboring columns of helices, as measured by P-P distances less than 8.65 A and by close van der Waals contacts between individual atoms, usually phosphate oxygen atoms and backbone atoms. As is customary, bases and sugars in strand 1 are labeled Cl to GlO and 81 to SlO, with

Cl 1 to G20 and 811 to S20 in strand 2. Phosphate groups are labeled P2 to PlO and PI2 to P20 in the two st,rands, numbered according to the base t,hat follows. Close van der Waals cont’acts are shown by dotted lines in Figures 5 and 6. Table 2 lists water bridges between helix columns. These two Tables should be compared with Table II of Grzeskowiak et al. (1991) for the KK helix. With the TA structure as with KK, the closest contacts between helices occur along the a direction of the unit cell (Fig. 5). A very tight contact involves backbone and phosphate oxygen atoms of residues 3 and 4 of the central helix shown in Figure 5, with similar atoms of residue 17 to the left. A somewhat less tight contact to the helix at the right, involves residues 8 and 9 of one helix with 12 and 13 of the ot,her. Along the b direction of the unit cell (Table 1 and Fig. 6), phosphate groups P6 and

Table 1 ( ’10s~ contacts

brtuwrr~

wlurrms

of h&us

in (‘-(l-.-I

-T-T-.4

-,3 -7’4 ‘4; crystals

----

,‘L 1’

Right

(.%I

1’17 I’IX PI7 1’18 Pi

Along h axis (Pig. 6) 13asr

AtOm

Hase

Atol”

c97 . .i..iO

a.;-,9 609 6% I

Along n axis (Fig. 5) 1)istanw

(A)

liase

:\tortt

IGMk

L~t~On’

I)istarrce(Aj

Al7

O-4'

.A3

O-II'

3.58

Ati

O-II'

(‘I I

(‘-.i’

346

Ali

O-3’

A3

0-11’

3~55

('X'

378

O-3'

A3

O-5

3X!l

Al i A 17 Ali TX ‘l-8 TX TX TX (‘9 ( ‘0

O-5 O-21’ O-.5’ o-IP o-%P o-%P O-3’ O-5 o-IP o-2P

A3 O-3 T4 0-IP ‘l-4 o-2P Cl% O-4’ A13 O-I I’ A13 C.5’ Gl% (‘-5’ G14 o-3 (:I2 (‘-5 GI2 O-3

O-21’ O-II’ O-3’ 0-J. I O-21 o-2P (‘-5 (‘3

(‘II

Ali

:i(i Ati (:%o c:20 Al6 Al6 (‘1 (‘I

(‘II A6 At?’ (:I0 c:10

(‘~3 P o-1 I’ O-3’ (‘-3’ O-‘IP O-21’

344 353 3-W z’T3 3.38 330 3%

3.44 3X5 351 35 I 387 :pp 36.5 38 I :wM 3X1

Al6

Altl

(‘ontavts are listed from left to right as drawn in Figs 5 and 6. and are listed from t,hr top of rdr drawing to the bottom.

Pl6 of each helix interact with the 5’ and 3’ ends of neighbor helices in the crystal. This efficient packing along the n and h directions of the unit cell is stabilized even further by ordered water molecules t)hat form hydrogen bonds between phosphate and sugar oxygen at’oms in two neighbor helices (Table 2). As Figure 4 illustrates, this interlocking of sugarphosphate backbones along the n direction holds the helices together in a manner that creates endless minor groove channels through the crystal in the ar plane. (See also Fig. 2 of Grzeskowiak et nl.. 1991.) (b) Magnesium

iw

bridging

betuwm h,eEices

Still another component of bridging between helices in the crystal is the Mg(H,O)i+ complex. At the upper left of Figure 5. the hydrated magnesium complex. if drawn, would be situated between backbone chains at the level of the label 16, sitting in the minor groove of the leftmost’ helix. This magnesium site is shown in close-up in Figure 7. looking down on the site from the rear as seen in Figure 5. Of the six water molecules coordinating the metal ion. two make hydrogen bonds with N-3 of A 17 (2.86 A) and O-2 of T5 (2.49 A) in the left helix as drawn in Figures 5 and 7 (thin tilled bonds). while t’wo other cxo-ordinating water molecules make hydrogen

bonds wit,h O-l P (2.7 I .A) and O-21’ (2.70 A) of’ X3 in t’he helix to the right (open bonds). The presence of the magnesium ion at this especally tight, contact near A17; but not at, the less tightly packed sequentially equivalent position near t,he sugar ring of A7. may contribute to t’he deformation observed in the Al7 sugar ring. As with the KK helix. this sugar adopts a (‘-3-credo conformation (ii = 90”) unlike all the other sugar rings. which are closer to (‘-%‘-mdo (107” I 6 I 155”). Torsion angle values of CY= 73”. fi = 233” and 7 = 196” at, Al7 suggest a compensating “crankshaft”’ rotation (Olson. 1982). The ctonformation of S17 and the torsion angles were tested by assigning them the same values as those found for t,he ~~qutw chain symmetrical S7 on the opposite tially (6 = 125, c1= 307”. fl = 146”. y = 196”) and refining the modified structure with NLTCLSQ. As found earlier with the KK helix, sugar conformation and torsion angles returned promptly to values similar to those observed previously. Restraining the Sl7 conformation to remain C-Zendo raised the refined R-factor from 1570/, to l6.6”/0 and led to peaks in the (PO- rC) difference electron density map of height 090 e~~/A’ (100) in the vicinity of S17. Thus. the S17 (‘-Kendo conformation is real, and reflects a deformation induced by tight packing of two

Structure

of a B-DNA

Decamer:

(r-G-A-T-T-il-A-T-C’-C

38.5

Figure 5. (Closeup of interaction of TA helices from the same viewpoint as Fig. 4 along the h axis. with the a axis horizontal and the c axis vertical. Lateral intermolecular van drr Waals contacts along the a axis are dotted. and their lengths are given in Table I. Nucleotides identified for reference are 16 and 18 in the left helix, 3 and 8 in the center. and 13 at the right. Adjacent helices run vertically in opposite directions. The central helix is related to that at its right by a horizontal 21 screw axis in the plane of the drawing, between the 2nd and 3rd base-pairs from the bottom of the central helix. Kote that contacts at the 2 crystallographically distinct ends of the central helix are similar in general but different in detail. The 3rd base-pair from the bottom of the cent.ral helix is at. the level of base-pair (‘9.(:12 at the right. If one whereas the 3rd pair from the top is at the level of pair Al 7 .T4 at the left, rather than the expect.ed (‘19.62. regards this sheet of helices in the UC plane as being built from vertical columns, adjacent columns are displaced slightly along their axes relative to one another.

helices: probably stabilized Mg(H,O)g+ cxomplex.

by the presence

of the

(c) Hydration, minor groove width and phosphate conformation The plot of minor groove width in Figure 3 shows that TA and KK decamers are similar in their alternation of wide and narrow groove widths with a periodicity of five base-pairs. The minor groove is wide at the center of the helix and at the interhelix stacking junctions, and narrow in between. The earlier observation (Grzeskowiak et al.. 1991), that the minor groove is widest when B,, phosphate groups are opposed across the groove, remains valid. As Appendix Table Al shows, B,, phosphate groups with positive values for torsion angle difference (c-l) occur at P2 and P7 in one strand and P12 and PI7 in the other; that is, always at Y-R or R-R steps, never at R-Y. The groove width ladder for the KK helix in Figure 11 of diagram Grzeskowiak rf al. (1991) can apply just as well to

the TA helix. The similarity in groove width may be ascribed to these two helices having identical patterns of purines (R) and pyrimidines (Y), even though the specific identity of t)hese groups has been changed at the center. i-\ well-defined spine of hydration is seen in each of the two narrow regions of the minor groove (Fig. 8(a)). These and the hydrated magnesium atom were readily visible in the first difl%rence maps after refinement of DNA, and maintained the lowest, R values or temperature factors of all solvent molecules. The spine of hydration in narrow minor groove regions, with first-shell water molecules that bridge thymine O-2 and adenine X-3 atoms, and themselves are bridged by a second shell of hydration, seems to be one of the most constant aspects of R-DNA structure (Drew 8r Dickerson, 1981: Kopka et al., 1983; PrivP ct al.. 1991; Grzeskowiak et al., 1991). Wider regions of the TA minor groove show two irregular ribbons of hydration that tend to bridge base edge N or 0 and sugar O-4’ at,oms (Fig. 8(b)). as was tirst encoun-

Figure 6. Closeup of interactions of TA helices in a view down the a axis. with the 6 axis horizontal and the c axis vertical. Lateral intermolecular van der Waals contacts along the 6 axis are dotted, and t’heir lengths are given in Table 1. Nucleotides identitied for reference are 6 and 20 at the left. Il. 6. 16, 1 in the center. and 10 and I6 at the right. Adjacent helices run vertically in the same direction. and major and minor grooves occur at the same c level in all columns. Indeed. if stacked decamers were copnected into infinite columns. and if the differences between A. T and (: (’ base-pairs could be ignored, then adjacent columns would be identical and the repeat in t.hr b direction would br halved. This is why. in the low-resolution region of the F, data set. reflections with k odd are syst,ematically weaker t,han those with k even. The central helix is related to those to it,s right and left by vertical 2, screw axes between molrc~ulcs. Xotcb that intermolecular packing of helical columns is more regular in this bc plane: nucleotides 16 and 6 have c~losrl,v similar (although crystallographically distinct) lwal rnvironmrnts.

tered in the GA decamer (Privk et al.. 1987, 1991). The magnesium complex contributes one water molecule to each of the ribbons, and another of its water molecules anchors the end of the spine of hydration in the adjacent) narrow region of the groove. No such regular pattern of hydration is where water observed in the major groove: molecules make hydrogen bonds with many of the N and 0 acceptor atoms in the base edges without an apparent pattern.

(d) Local structure The

striking

in TA and KK

is that

observation

substitution

of one

with

helices

new (T) for another (C), and one purine (A) for another (G). at the center of the helix produces almost, no change in local helix structure. Parameter values for the TA helix in Tables Al to A3 of the Appendix are nearly identical with those for the KK helix in the analo-

decamer

most

variation

this

pyrimidine

gous tables of Grzeskowiak et al. (1991), so much so that it is not worth taking valuable space t’o repeat the plots. As a single representative example, Figure 9 shows the behavior of the Profile Sum for the two helices. As defined by Yanagi et al. (1991), the Profile Sum is a joint expression of the closely linked hase step variables twist (w), rise (II,), cup (x) and roll (p): Profile

Sum = (w - 36) - 1624(I), - 3.36) +0.744x -0.703p.

High Twist Profile (HTP) steps from one base-pair to another are observed to have high twist, low rise, positive cup and negative roll, and to have Profile Sum values of the order of + 10 or great,er. In Low Twist Profile (LTP) steps are contrast, characterized by low twist, high rise, negative cup and positive roll, with Profile Sums of - 10 or less. Tn the context of this sequence: T-A is an LTP step. But, just, as C-G steps were found to he highly

Structure

of a B-DNA

Decamer:

387

G-G-A-T-T-A-A-T-G-G

complex. The crystallographic a Figure 7. Bridging of 2 helices by a Mg(H,O)‘,’ base-pairs rising toward the viewer are A3.Tl8, T4.Al7 and T5.Al6. At the right, falling away from the viewer, are G2, A3 and T4. Hydrogen bonds between water acceptor atoms in the 2 symmetry-related helices are drawn with broken lines, while Waals contacts between the sugar-phosphate backbones of the 2 helices.

axis is horizontal.

dotted

lines indicate

(lup(n)

= Huckle(7l+

Positive cup occurs when the 2 base-pairs buckle as shown in Fig. 5 of Yanagi et al. (1991). like 2 cupped hands brought palm to palm. All X-ray crystal structure papers on synthetic DNA olipomers since the 1988 Cambridge nomenclature accords have used this sign convention for buckle).

3.50 A or lens)

betwPPn, columns

in

C-G-il-T-T-A-A-T-C-C

Atom 1

Distance Atom l-Water

ll’ater

no.

Distance Water -Atom 2

Atom 2

2.40 2439 2-74 2.84 2.62 2.65

24/05

3.34

Q12

O-21'

24/07 24/21 24j2ti

3.4 1 327 3.01

G20

O-3'

c:1 GlO

O-.5 O-3’

261

24/01

2.99 W9 2.93 3.23 296 2..55 “47

T18 A 17

Al3 c9 A3

O-IF'

3.39 3.3 1 2.86 2.99

A3 U2 TX A13

o-1P O-3’ O-I I’ O-11’

340 2.90 2.88 349

Cl 1 (‘11 Cl

CM’ O-5’ O-4

(‘1

O-.3’

283 3.15 3.01 3.18 3.18 307 2.87

24102 24/09 24114 24/16 24123

3.23 3.56

22/14 24/13

(‘. Retw~en htdiees in one column c-20 r N-3 2%:~ ( :20 O-4 2.51 (:I() N-3 3.14

close van der

1) - Kuckle( n).

Table 2 (distances

the

the diagram. This is an intuitively reasonable sign convention, since the concept of buckling suggests a doming upward of a pavement or other surface. If the sign convention of buckle were to be arbitrarily inverted, the variable itself would be more properly of buckle. With the instead called slump KEWHEL91 sign convention for buckle, cup is defined as the difference between a given buckle and that of the base-pair that preceded it:

variable, so the evidence of all the other B-DPI;A decamers and dodecamers taken together establishes that T-A steps must also be classified as VTP (Variable Twist Profile), strongly susceptible to influence by their environment. (Some confusion has developed recently over the sign of buckle, and therefore of the definition of cup. As defined by the 1988 Cambridge nomenclature accords (Dickerson et al., 1989), strand 1 of the double helix is taken as rising upward along the z axis. In Fig. 8(b), base-pair Cl * G20 is at the bottom and GlO.Cll at the top. Buckle, as calculated by NEWHEL91, is positive when the base-pair domes convexly in the 5’.to-3’ “forward” direction of strand 1. Hence the 2nd base-pair from the bottom in Fig. 8(b) has a negative buckle (-14.1”): the center of the base-pair sags toward the bottom of

Water bridges

At the left,

3 bases from a neighboring helix, molecules of the Mg complex and

25121

22/11 22/ 10

1’18 (:2

O-II’ ( )-3' O-11’ O-11’

O-11’ o-11’

(a)

0

0

(b)

Figure 8. Stereo views of the TA decamer showing minor groove hydration. Crossed spheres indicate water oxygen atoms or magnesium ions. Interatomic distances of 3.5 A or less are drawn as thin lines. The vertical axis drawn is the best overall helical axis through all 10 base-pairs. (a) Two spines of water molecules run along the top and bottom parts in the narrow regions of the minor groove. (b) A Mg(H,O)z+ complex in the lower half of the helix is located at the end of 1 spine of hydration and leads to a wider region of the minor groove with 2 ribbons of water molecules not as orderly arranged as the spines. No Mg(H,O)g+ complex was located in the sequentially symmetric related region of the minor

groove. In this drawing, base-pair Cl -G20 is at the bottom and GIO.CI 1 is at the top, although virtually of identifying

the 2 ends visually

(e) Base-pair

are the presence

of the magnesium

stacking

Stacking of the ten individual TA decamer (including the between helices) is shown in

base steps for the non-bonded step Figure IO. These

ion and the aberrant

the ouly means

sugar conformation

at A17.

diagrams are astonishingly similar to those for the KK helix in Figure 12 of Grzeskowiak et al. (1991), not only at the ends of the helix but in the center as well. In particular, steps (a) to (c) and (h) to (j) in the two Figures are distinguishable only by very

Structure of a B-DNA

and observed the following behavior for base steps:

IOO-lO-2o-3o-401:t 012345678

9

IO

II

Base step Figure 9. Profile Sums at each of the 10 steps of stacked TA decamers. Step 10 at the right is unbonded step between stacked helices. Filled circles heavy line, TA helix. Open circles and light line,

the the and KK helix. All 3 of the C-G steps in the KK helix are LTP (Low Twist Profile). In the TA helix, the central T-A step mimics C-G precisely. T-A, C-G and C-A steps all must be

considered as variable, from

their

general

twist,

profile

HTP: G-C and G-A (= T-C) ITP: A-T and A-C ( = G-T) T,TP: A-G (= C-T), A-A (= T-T) and G-G (= C-C) VTP: C-G and C-A ( = T-G).

20E G al = ‘;; Lr

389

Decamer: C-G-A-T-T-A-A-T-C-G

especially susceptible to influence

local environment.

close inspection of fine details. In general, R-Y steps such as A-T are characterized by overlap of the sixmembered rings of both purines and pyrimidines. R-R steps pivot about the six-membered rings of purines, so that the O-2 of the 5’ pyrimidine sits over the center of the six-membered ring of the 3’ pyrimidine, a stacking mode that was noticed and commented upon by Heinemann & Alings (1989). Y-R steps, however, exhibit virtually zero overlap of base rings, no matter whether the step is LTP as in T5-A6 of this TA decamer or HTP as at C2-A3 of the CG and HA decamers. The central steps of the two helices, at (e) in both sets of stereos, are distinguishable most notably only by the difference in base-pairs, A. T versus G. C. The presence or absence of the minor groove C2 amine appears to have no effect at all on basepair stacking. B,, phosphate conformations occur at the same positions in TA and KK structures, at the right in Figure 10(a) and (f), and at the left in (e) and (i) and, as has been noted earlier, produce the same effect on groove width. The unusual, crystal packing induced C-3’-endo conformation of the sugar of Al7 is seen at the left in Figure 10(c) and (d).

4. Discussion Travers & Klug (1987, 1990) have reviewed the experimental evidence supporting the particularly large instability, deformability and conformational flexibility of the T-A step in solution. This paper adds another link to that chain of evidence, by showing how the T-A step can adopt different conformations in different sequence and crystal packing environments. Grzeskowiak et al. (1991) surveyed the crystal structure analyses of 12 R-DNA oligomers (4 decamers and 8 dodecamers)

In that, study, T-A was included as an HTP step solely on the basis of the Yoon dodecamer crystal structure analysis (Yoon et al., 1988). Now we must reclassify T-A steps as having a variable twist profile (VTP), depending on the local environment. A standard manner of displaying the 16 two-base steps is a 4 x 4 matrix with bases arranged as in Figure 11, first base down the left side and second base across the top. With this particular G-A-T-C order of bases, self-complementary steps occur along the principal diagonal of the matrix, from lower left to upper right, and pairs of complementary steps such as G-T and A-C are arranged symmetrically across this diagonal. The set of unique steps in a double helix then consists of all of the diagonal terms, plus one group or the other of the offdiagonal terms, or 4+6 = 10 of the 16 steps. If the observed twist profile behavior is mapped onto this matrix, the result is Figure 11. This mode of display illustrates a relationship that was unnoticed previously: R-R steps all are LTP, with one HTP exception: R-Y steps are all ITP, with one HTP exception: Y-R steps are all VTP,

G-A (= T-C) G-C’

These three relationships, including the exceptions that define the HTP class, must be a consequence of the energetics of base-pair stacking, although these relationships are not obvious yet. This matrix may change as more oligomers are studied, but it reflects what has been seen to date in crystal structures of dodecamers and decamers of B-DNAt. The T-A step in DNA has been the subject of many different biophysical studies. Klug et al. (1979) predicted an alternating co-polymer model for poly(dA-dT) based on the crystal structure of the dimer p-A-T-A-T (Visvamitra et al., 1978). In this “alternating-R” helix, the intrinsically badly stacked T-A steps destack even further (thus t One referee commented upon the similarity between our Fig. 11 and Table 1 of Calladine & Drew (1984). The 2 tables, however, convey quite different information. The 1984 table expressed the particular bi-stability of Y-R (pyrimidine -purine) steps between A and B-helices; the tendancy of such steps in the 2 helix types to occupy different and separated regions on the Roll/Slide conformational plane. In contrast, our table describes the ability of a Y-R step to exhibit at least 2 different twist profile behaviors, both within the B helix. (A corresponding analysis of A-DNA has not been made, primarily because until recently relatively few A-DNA co-ordinate sets were in the public domain.) The only feature common to both tables is that they describe something unusual about Y-R steps, which itself is the subject of an extensive literature.

390

J. M. Quintana

(b)

(d)

Fig.

10.

et al

Structure

qf a

B-DNA

Decamer: (!-G-A-T-T-A-A-T-C-G

391

(h)

(i

1

(j I Figure 10. The 10 independent base-pair steps of the C-G-A-T-T-A-A-T-C-G crystal structure, including the nonbonded step from GlO.Cll at the top of one stacked helix, to Cl .G20 at the bottom of the next. A small dot near the center of each drawing marks the best overall helix axis. Twist profiles and right/left phosphate conformations at the 10 steps are: (a) Cl-G2: ITP, II/I; (b) G2-A3: HTP, I/I; (c) A3-T4: ITP, I/I; (d) T4-T5: HTP, I/II; (e) T5-A6: LTP, I/I; (f) A6-A7: HTP, II/I; (g) ,47-T& ITP, I/I; (h) TS-C9: HTP, I/I; (i) C9-GlO: ITP, I/II; (j) GlO-Cl: LTP. I and II indicate B, and B,, phosphate groups. HTP, ITP, LTP, High, Intermediate and Low Twist Profiles. Compare Fig. 12 of Grzeskowiak rt al. (1991).

G A T c

twist to avoid sterir clash bc%wtaen ath~ninc~ bases from opposing strands. Theoreticaal c.al~la Cons by (Ihuprina (1987) indicate t,hai the spine of’ hydration observed in the narrow minor groo~t~ 01 adenine t.racts of several DNA dodecamcr c*rystals (Drew & Dickerson. 1981; Kopka rt al.. 1983) would be energetically unfavorahh~ were a T-A step to bcl introduced, and that a wider minor groovfb would be the consequence. These c?onsiderations t akthn t,ogether would lead one t,o expect both a high twist and a wide minor groove at a T-A sttlp. The present, TA decamer c*ryst,al st,ruct,ure would appear to support the wide groove portion of’ tht, above argument. One can regard the cent,ral region of C-C:-A-T-T-ADA-T-(I-(: as a disruption in t)hc middle of a six base-pair A .T trac>t. just as thtk propeller

GATC LH L L v v v v

i i L L

H i H L

Figure 11. Summary of Profile Sum behavior of basepair steps from dodecamer and decamer H-DNA X-ray crystal structure analyses of synthetic oligonucleotides. H, HTP; L, LTP; i. TTP (Intermediate Twist Profile). v. VTP (Variable Twist Profile). The 1st base of the step is at the left, the 2nd along the top. Note that all Y-R OI pyrimidine-purine steps are VTP, and are more sensitive to their environment than are R-Y or R-R (= Y-Y) steps. R-R steps tend to be LTP, with a notable exception, the C-A step. R-Y steps tend to be ITP, again with 1 excrption. G-C’. Both of these exceptions tend to be HTP.

HTP) in order to improve stacking at the becoming inherently more overlapped A-T steps, giving them LTP character. This prediction was confirmed subsequently by a crystal structure of the dodecamer (1-G-C-A-T-A-T-A-T-G-C:-G (Yoon rt al., 1988), which exhibited a regular alternation of t,wist, between A-T and T-A steps. The T-A step has been shown by gel electrophoresis and nuclear magnetic resonance measurements to interfere with DNA bending when of A. T base-pairs introduced into a region containing only A-A and A-T steps (Koo rt al., 1986; Hagerman, 1986; Leroy et al., 1988). From observation of hydroxyl radical cutting frequencies, Rurkhoff & Tullius (1988) propose that the structural change responsible for the avoidance of bending is a widening of the minor groove at’ T-A steps. Nuclease digestion studies by Drew & Travers (1984) also predict widening of the minor groove at T-A steps, possibly because of a damping down of

center

of the

KK

helix,

(“-(:-A-‘I’~(‘-C~A-‘I’~(‘-(:.

Table 3 Helix

Helix KK TA Am&t C‘Oll Balm. Y 0011 Yoon

KK TA (‘011 Balen. Yom

parameters

of T-A

steps and a comparison

C-G step

SW

Twist (deg.)

Rise (A)

Cup (deg.)

Roll (deg.)

c5-G6 T5-A6 Standard T6-A7 T6-A7 T5-A6 T7-A8

293 31.1 360 396 42.5 39.5 43.2

4.08 3.98 3.36 2.61 2.84 2.75 3.37

-4.4 ~ IO.6 0 + 11.1 + 10.1 f21.6 +4,5

+ 8.5 + !I4 0 -. 1.7 +4.4 i-l.4 + 8.X

= (‘-G-A-T-C-G-A-T-(:-C: = (‘.(~.A.T.T~~-A-1’-(‘-(: = (~:-C:-C:-G-A-T-A-T-(‘-C:-(:-(: = (I-(:-(‘-O-T-T-A-4-(‘-(:-(‘-(: = (‘-(:-C~A~T~A~T~A-T-C:-(‘-(:

+ Netropsin

(WI

be so regarded. In the minor groove width plot of’ Figure 3. the intrusion of a C’-G or a T-,L\ st,ep ma) bra taken as responsible for t.he appearanc*c of’ t,hfl cbentral peak in what otherwise would he a six bastepair narrow groove region. But thta rxpr4ed t)wist behavior is not observed: the central step, whether (!-G or T-A, is LTI’ rather than t,hr expec%rd HTI’. Perversely. the (‘-C:-(‘-ANT-A~‘~-A-‘r-(:-( ‘-G strut t)ure described b? Yoon P’I crl. (1988) rontradic*ts both of these findings. In that structure. the minor groove remains narrow all along the six A T bastxpairs. yet the T-A steps are HTP. Henc*e. as has been not,ed hy (~alladine & Drew (19X4) and ot)hrr.s, a T-A step must be inherently variable. c~apablc of adopting at least. two different c~onforrnatiorls. ill t ht* present insta,nc*cA, leading t.o t1it’ht.r witl(, or narrow minor grooves. and to large or small hrlicaal twist Furt,hermore. helical twist and minor groove width are not inevitably coupled. as frequently has been assumed. The stacking behavior of T-A steps in these different, B-DNA crystal environments is compared in Figure 12 and the values of local parameters and Profile Sums for several T-A steps arr listed in Tahlr 3. The concept of the int’rinsic weakness of’ stacking

(Grzeskowiak et nl., 1991) (This work) (Co11et ml., 1989) (lialendran, 1991) (Yoon Pt 01.. I988)

Profilr Sum - 27.6 -dT+i 0 + 258 + 26.9 f264 + 4.2

Structure

qf a

R-DNA

Decamer:

(‘-G-A-T-T-rl-A-T-C1-C:

393

(b)

(9) Figure 12. Variability of twist profile of the T-A step in 3 different, DNA structures: (a) C&G6 step from the center of the KK decamer: o = 293’, PS = -28, LTP; (b) T5A6 step from the center of our TA decamer: o = 31.1”. PS = -28. LTP; (c) ideal T-A step from fiber co-ordinates: w = 36”, PS = 0. TTP (Chandrasekaran & Arnott, 1989): (d) T&A6 from the alternating dodecamer C-G-C-A-T-A-T-A-T-G-C-G: o = 38.9”, PS = +26. HTP (Yoon it al.. 198X): (P) T’7-A8 from the same alternating dodecamer: w = 41.1”. PS = +4, HTP. PS. Profile Sum.

at a pyrimidine-purine step is reinforced by the observation that base-rings in all three types of Y-R step, C-G, C-A and T-A, overlap virtually not at all, regardless of whether the step is high or low twist,. Grzeskowiak et al. (1991) observed that one feature

of the VTP steps C-G and C-A was their susceptibility to influence by flanking steps, the essence of the tetrad hypothesis. If the adjacent steps were HTP or ITP, the central VTP step would be pushed int,o a low-twist, mode as in the tetrad (X-A-T; if

wither of the flanking steps was H’rl’. thrrl t,tw writ ral step could open up as in (‘4 ‘-A-A. f’q. this reasoning one would expect HTJ’ behavior for t,he central T-A step of the decamer c~-(:-,-\-‘r-r--\-i\-‘r( ‘4:. since both of t,he flanking steps are predicted t,o tjtb f,Tf’. f%ut t,his is not t’he case experimentally: Figure 9 shows that, t,he t,hree steps of T-T-A-A in t 1~~ TA drcamclr t,xhibit precisely thr invrrstb IJrhavior: H-Id-H. The expected I,-H-l, twist profile behavior of the ‘r-‘r-i-\-A tetrad is found in a recent structure analy~ sis of’ another dodecamer, (:-(:-(‘-~;-‘r-T-A-A-(‘-(:( ‘4 : ( Kalendran. 1991, and personal comm uncation). This dodecamer st,ructurr is isomorphous with that of the I)rew dodrcamer (‘-(:-(‘-(:-A-A-‘rT-(‘-(:-(1-C:. and indeed with all of the other dodrc:a rnrr structures (see Fig. 2 of Dickerson of ~1.. 1987). Alt,hough the rx~jected twist behavior is seen. thta narrow minor groovr still is not widened t)y the T-A step. Hence the t,etrad hypothesis. alt,hough clearly an improvement over earlier attempts to characterize base steps in isolation, still is not the entire story. One and the same tetrad, T-T-A-A. can adopt different conformations in different environments. Not all tet,rads will be equally malleable; thosck involving the central T-A step srrm to he especialI>, .‘soft “1 The aforementioned lack of iJa,se-pair overlap in T-A sttp tnay he the root cause of this hrhavior: t)heoretical st,acking energy calculations ohviousl!- are needed. To reiterate a fundamental c*onc:lusion st,ated earlier: (Irystal structure analyses of B-I)KA oligomers have shown t,hat the T-A step in it double helix can have either high t)wist, or Ion twist. and can participate in either a narrow or a wide minor groove. Furthermore. t.wist) angles and minor groove width are not, coupled. The DNA double helix is neither H rigid. undo formable rod. nor a shapeless st’riny of spaghetti. (‘rrt.ain regions of sequence appear to have a reasotlahI!- well-detined structure, whereas other regions are susceptible to deformation by the environment in defined ways. One is driven t,o what could he termed the articulat’ed helix model of DXA strucature. A length of DISA ca.n be compared with a human arm. The real arm is not. represented fairly [J>- a static sculpture: neither is it a floppy garden hose. Some parts of the arm such as the wrist can both bend and twist. Ot,her parts such as the elbow can bend, but, not twist. St.ill ot,her part*s such as the forearm char1neit,her bend nor twist. A person who is designing tasks for t,he arm t’o carry out had better know rat,her accurately what is possible for each region of the arm. A given DNA base sequence chooses a particular csrystal packing mode because t,he constraints of that mode are compatible with it’s own possible motions. Tn the previous analogy. the monoclinic8 and orthorhombic packing modes can be likened to left and right-handed gloves. No one would ever claim that a glove induces the structure of the hand that fits into it. but by the same token one will not expect to find a right hand fitSting int.o a left, glove.

SO t’o 1he question. ‘~f)Of’h tlO~~iJl~~-t~~~li~~ill1)X21 have orw single. rigid, sec~ut~rlc~r-inci~~(~~~~it ru(’ ture 1”. one must reply. “Y1 o .’ f3ut thcs ciur~stiori. “1> the strucsture of DKA as viewed in t hc (~r~siallo graphic analysis determined IJ~ carystal IJa(~kitlg!“. A giver1 c~r.yst.ai receives t,he same answer. “No”. st,ructurr analysis t)raps orw of t htl c~onformat ions that are itvailatjle to that partic~ular stquc’trl3~ of l)NA. As one coniparw vitlues of’ pitranic~ljc~rh of similar regions of DIVA iri cliffererit st~rrlc~turt~~. on(~ begins to IJuild up a knowlrdgt~ of’ I ht. t ariou~ conformations that are open to t,hat part itarrlar sequence. Eventually we shall l~r able to tlesc~riht~ irj detail the anatomv and kinesiology of o11r (1011l~lt~ helical 1)N.A .‘arm”‘. t)ut that time is not >.(*I !icar~,.

Appendix Table Al Torsion

a,ngles for tlw 7’A derumPr

Table A3 Haxe-pair

parameters

for

the

TA decuwrrr

Structure

of

a B-DNA

Decamer: (‘-C-A-T-T-A-A-T-C-G

395

B-DNA sequence

its J.

We thank Dr Hanna Yuan for discussions on DNA conformation, in connection with her closely related C-GA-T-A-T-A-T-C-G sequence. This work was supported by National Science Foundat,ion grant DMB85-01682 and National Institutes of Health Program Project grant GM-31299.

10, 35-43.

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by A.

Klwg