Sequence-dependent effects in A-DNA double helices

Journal of Molecular Structure (Theochem), 179 (1988) 367-391 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

SEQUENCE-DEPENDENT HELICES* TALI E. HARAN and ZIPPORA

367

EFFECTS IN A-DNA DOUBLE

SHAKKED**

Department of Structural Chemistry, Weizmann Institute of Science, Rehovot 76100 (Israel) (Received 7 March 1988)

ABSTRACT To date, more than ten different A-DNA structures have been analyzed at near atomic resolution by X-ray methods. This structural information enables us for the first time, to begin to discover the rules which govern the conformation of the double helix in the A form. A detailed analysis of the various helical conformations show that local structural changes are induced by base-pair stacking interactions. Variations in stacking geometry are sequence dependent. For pyrimidine-purine sites, the stacking mode is determined by the nature of the base-pair doublet only, whereas for purine-pyrimidine and homopolymer sites the identity of neighboring base pairs is important as well.

INTRODUCTION

‘Twas brillig, and the slithy toves Did gyre and gimble in the wabe: All mimsy were the borogoves, And the mome raths outgrabe. “It seems very pretty”, she said when she had finished it, “but it is rather hard to understand!“... “Somehow it seems to fill my head with ideas - only I don’t exactly know what they are!” (From “The Jabberwocky”,

in “Through

the Looking Glass”, by L. Carroll.)

Genetic material has often been compared to a language, the language in which Nature transfers messages and information for future generations. Proceeding with this analogy, the letters are the four building blocks, the nucleotides or only the bases. Words are short runs of nucleotides, for example, those specifying promoters, operators, or recognition sites of restriction enzymes, and the sentences are complete operons, or the individual genes. It has been a while since the old paradigm of a regular, monotonous, and *Dedicated to Professor Bernard Pullman. **To whom reprint requests should be addressed.

0166-1280/88/$03.50

0 1988 Elsevier Science Publishers

B.V.

repeating structure for the DNA double helix was replaced by the idea of a sequence-dependent and flexible structure for the DNA double helix. It has been shown by various groups that the DNA double helix does not have a regular and monotonous structure, but has conformational variations in the structure, determined by the specific sequence of the bases in a given region. Nevertheless, until recently our understanding of the vocabulary of this language could be likened to our understanding of this nonsense rhyme by Lewis Carroll. We knew the letters and some of the more simple words, but we did not know the rules for combining them into meaningful sentences. X-ray analyses of single crystals of deoxyoligonucleotides carried out in the past few years yielded detailed structural information on three forms: two righthanded structures similar in their overall conformations to the well established A- and B-DNA double helices derived by fiber methods (see reviews by Shakked and Kennard [ 1 ] and Dickerson et al. [ 2]), and a radically new left-handed double-helical structure named Z-DNA and observed mainly in alternating sequences of purine and pyrimidine bases (see review by Wang and Rich [ 31) . The available data on the left-handed helices indicate that the Z-DNA backbone is highly rigid and only slightly affected by changes in the base sequence. In contrast, structural variability within the various fragments of the righthanded forms is relatively large and sequence-dependent (see review by Shakked and Rabinovich [ 41) . Most of the data presently available is for the A form of DNA. More than ten different sequences ranging from a tetramer to a decamer were shown to adopt the A-type double helix whereas only few oligonucleotides were crystallized as far as B-type double helices. In addition to these, several mismatchcontaining duplexes related to those analyzed by Watson and Crick were investigated [ 51. In this article we deal mainly with the Watson-Crick-type helices of the A form and examine the effect of the base sequence on the fine structure of the double helix in terms of conformational features and energy considerations. We believe that the study outlined below is another step in establishing our dictionary to the sequence/structure vocabulary of the language that the DNA molecules prefer to speak. RESULTS AND DISCUSSION

Global helical conformations Average helix parameters for A-DNA structures, determined by X-ray diffraction methods, are given in Table 1. Definition of the various parameters are given in Fig. 1. The A-duplexes have common features typical of the A-DNA form deduced from fiber diffraction. As shown by Table 1 and also noted before [ 1,6], the

369 TABLE 1 Average helix parameters Compound

Ref.

Helix

twist (“)

d(GGTATACC) d(GGGGCCCC) d(‘CCGG)/d(‘CCGG) d(GGCCGGCC)(-8°C) d(GGCCGGCC) d(CCCCGGGG) d(GGGCGCCC) d(GGGTACCC) r(GCG)d(TATACGC)

15 16 6 17 10 8 9 18 19

Rise/ residue (A)

Bae pair tilt (“)

31.9 31.6 34.0 32.6 32.9 33.5 31.5 30.0 33.3

2.92 2.94 2.84 3.00 3.09 3.08 3.26 2.88 2.52

13.2 12.6 14.0 11.9 11.4 9.0 6.5 13.2 19.4

Mean

32(l)

2.9(.2)

12(3)

A-DNA’ B-DNA’

32.7 36.0

2.56 3.38

22.0 -2.4

Groove width M_ajorb

Propeller D,” twist (A) (“)

(A)

Minor (A)

6.3 6.8 4.8 7.9 7.7 8.6 10.1 8.2 3.2

10.2 10.2 10.6 9.6 9.8 9.4 9.4 9.7 10.2

10.2 10.5 16.3 8.6 12.5 10.8 10.0 9.4 11.8

4.0 4.2 3.7 3.6 3.4 3.9 3.8 4.6 4.5

10(.4)

11(2)

4.0(4)

11.2 7.4

6.0 13.3

7(2) 3.7 14.7

4.4 -0.6

“Distance of C6-C8 vectors from helix axis. bEstimated from the distance between terminal 5’ -phosphorous atoms. ‘Based on fiber data of Amott and Chandrasekaran (unpublished).

most important feature, differentiating the A family from the B one, is the positioning of the base pairs, as reflected for example by the values of D, the distance of the C6-C8 vectors from the helix axis. The base pairs are positioned on the helix axis for B-DNA, but pushed away from the axis in the direction of the minor groove for A-DNA. This characteristic can be used to classify newly determined structures. The detailed structures of these helices differ from the canonical 11-fold A-DNA fiber model, and also from one another, as seen from the values of the various parameters given in Table 1. The average helix rotation or helix twist is the only parameter having similar values in all A-DNA structures, and displaying the least departure from the fiber-derived values. It has been observed both in fibers and in the single-crystal studies that the variations in the average height, or rise per base pair, are anticorrelated with the average values for the base-pair tilt [ 1,7]. As the tilt increases, the rise per base pair decreases. A wide range of values is observed for the major-groove width of the A-DNA octamers (Table 1) . The major groove width is correlated with the rise per base pair and anticorrelated with the base-pair tilt. The correlation coefficient derived from nine A-DNA crystal structures is 0.98( 10) for the rise/base-tilt relation and 0.92 (20) for the rise/major-groove one (Figs. 2 and 3). Fiber diffraction studies also show that the major groove widens as the rise increases and the base tilt decreases [ 71. Much smaller variations are observed for the minor-groove width of the various A-DNA helices, compared to those for the major-groove width. Such behavior may be visualized by imag-

Helix axis

f

(a)

Major groove Twist

Helix axis (vertical)

Roll

+

Tilt

Minor groove

(b)

Helix

axis(vertical)

Fig. 1. The local axial system used to define base-pair position and orientation: propeller twist is the dihedral angle betwen the two bases of a pair viewed along its long axis (a). Helix twist is a rotation about the helix axis (b). Local twist values are estimated by the angles between successive interstrand Cl ’ -Cl ’ vectors projected down a plane normal to the global helix axis. Roll and tilt angles between base pairs (Sa,&) define the change in orientation from one base-pair to the next one and are independent of the helix axis. They measure the angle by which the two base pairs open towards the helical groove or towards the backbone, respectively (b). Slide (S) measures the relative displacement of adjacent base pairs along their long axes. It is estimated from the distance between the midpoints of two successive interstrand CG-C6 vectors projected onto the vector connecting the midpoints of the two intrastrand ones (c). Analytical definitions of the various parameters wem given by Fratini et al. [ 201 and in the Appendix by Dickerson et al. [ 21.

371

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8

9

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15

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Fig. 2. Correlation plot of rise and base-pair tilt in A-DNA structures. The correlation coefficient is -0.98(10).

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3”

”

4

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5

” MA,:,

n ” ” 7 8 GROOVE WIDTH (ii)

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Fig. 3. Correlation plot of rise and major groove width in A-DNA structures. The correlation coefficient is 0.92 (20).

ining a double-stranded ribbon wound around a central core with the minorgroove side of the ribbon facing outwards; in this manner one would expect the minor-groove width to remain invariant and the major-groove width to be dependent on the way in which the ribbon is wrapped around this core. Elongated helices derived from d (CCCCGGGG) and r (GCG)d (TATACGC) which exhibit two extreme conformations of the A-type helix were shown by Haran et al. [8]. In the first structure the base tilt is small, the rise per base pair is large and consequently the major groove is wide. In the second structure the base tilt is large, the rise is small and hence the major groove is narrow. Jain et al. [23] have observed a correlation between the base-pair tilt and the base-pair displacement (D); the correlation coefficient based on 28 pairs from five A-DNA structures being 0.81. The major contributions to this relationship come from the extreme values displayed by the octamer d (GTGTACAC ) and the hybrid decamer. The average values for base-pair tilt and base-pair displacement are 9.3” and 3.1. A for the former and 19” and 4.5 A for the latter. It appears that a large tilt may be associated with a large displacement because as pointed out by Jain et al. steric clashes are caused when the base pairs are highly tilted and close to the helix axis. However, as shown by the average values of d (CCCCGGGG) and d (GGGCGCCC ) (Table 1) , a low tilt does not necessarily involve a small displacement. A large variance in D values (3.4-4.6 A) is observed for duplexes with moderate tilt values (ll13” ). The specific values are probably affected by the base sequence and hydration. Torsion angles and sugar-phosphate backbone conformation The average backbone conformation in all A-DNA fragments determined so far are similar to each other, and to the 11-fold A-DNA and A-RNA double helices deduced from fiber diffraction studies (Table 2). The definition of the various angles is given in Fig. 4. The torsion angles at the 5’ side of the sugar ring show the largest variability in every A-DNA duplex. a and y torsion angles exhibit the highest flexibility. These are also the angles that are most significantly correlated (Fig. 5). Since the angle /3 is trans in all these structures, the flanking bonds are antiparallel, and hence can be rotated with respect to each other in a crankshaft motion. In other words, when one angle increases the other decreases. The conformation about these angles is usually (A, in Figs. 5 and 6(a) ). gauche -gauche + as deduced from fiber diffraction The crankshaft-type motion brings about large changes in a! and y torsion angles associated with C-G base-pair doublets. An extended backbone, with (Y,/3 and y all displaying the trans conformation, has been observed at the CG base pair step of d(CCCCGGGG) [8], d(GGGCGCCC) [9], d(GGCCGGCC) [10],d(GGm5CCGGCC) [ll] andd(ACCGGCCGGT) [12]. This is represented by AI1in Figs. 6 and 6 (b). The extended backbone appears

373 TABLE 2 Average torsion angles ( o )

d(GGTATACC) d(GGGGCCCC ) d(‘CCGG) d(GGCCGGCC) (-8°C)” d(GGCCGGCQb d(GGmSCCGGCC)b d(CCCCGGGG)b d(GGGCGCCC)b d( GGGTACCC ) r(GCG)d(TATACGC) A-DNA” A-RNA’ B-DNA” B-dodecamer

a

B

Y

-62(12) -76(14) -73(4) -75(36) -67(S) -60(29) -87(11) -73(12) -67(22) -69(31) -50 -68 -33 -63(8)

163(8) 178(10) 180(S) 185(13) 175(20) 172(22) 182(13,) 177(S) 169(15) 175(14) 172 178 138 171(14)

52(14) 63(14) 64(10) 56(22) 68(26) 51(19) 72(7) 63(12) 16S(19) 55(22) 41 54 33 54(8)

f3 88(3) 84(11) 80(6) Sl(18) 81(4) 83(11) 7S(2) 81(11) 78(5) 82(S) 79 82 142 123(21)

f

cc

X

-152(8) -156(13) -161(7) -166(19) -142(19) -159(18) -153(13) -157(11) -156(11) -151(15) - 146 -153 -141 -169(25)

-78(7) -70(19) -69(4) -75(19) -76(8) -70(16) -66(7) -68(14) -74(15) -75(16) -78 -71 - 157 -108(34)

-M(8) -158(10) -161(7) -149(10) -161(10) -160(10) -162(S) -163(10) -163(E) -162(10) - 154 - 158 -139 -117(14)

“Omitting the 6 values of disordered sugars of Gl and G2. bOmitting fifth nucleotide values for the (Y and y angles. ‘Based on fiber data of Arnott and Chandrasekaran (unpublished).

to facilitate the sliding motion between the two C-G base pairs in a direction that improves the cross-strand stacking between the guanine bases. A similar backbone conformation was proposed by Crick and Watson for the first BDNA helix [ 131. Based on semiempirical potential energy calculations, Olson [ 141 have proposed the a/r correlation among other correlations in B-DNA helices. The (x/ y linkage appears however to be much more relevant to A-DNA-type structures [ 81. The changes in the two angles follow the allowed base stacking pathway described by Olson and cover the complete range from the gauche -,gauche + conformation to the trans, trans one and slightly beyond. More extreme values for this pair of angles are unlikely to be found because the change from the trans, trans conformation to the gauche +,gauche - one is accompanied by a decrease in the base separation distance to below van der Waals’ distance, leading to a sharp rise in the potential energy [ 141. Olson also suggested that the a/~ correlated movement is accompanied by a general opening of the helix, and by an increase in the base separation distance, as the angles change from the more common gauche -,gauche + state to the trans, trans one. This can be seen by the rise per residue values of the C-G base-pair steps in d(CCCCGGGG), d(GGCCGGCC) and d(GGGCGCCC) (3.3, 3.4 and 3.3 A respectively) and by the major-groove width of the structures (Table 1) . Other structural correlations in A-helices have also been observed [ 1,6,15]. Table 3 depicts the current state of these correlations, including all A-DNA single-crystal structures available to date. RI2 is the two-variable correlation coefficient and a(R) is its estimated error as defined in Table 3. A comparison

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I 120

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x 160

I p80

gouche-

trans

gauche+

(b)

Fig. 4. (a) The standard DNA backbone conformation showing main chain and glycosidic-bond angles. Torsion angles are labeled (Ytrhough < going in the 5 ’ to 3 ‘ direction from one P atom to the next. Glycosyl torsion angle (x) is defined by 04’-Cl’-Nl-C2 for pyrimidines and 04’-Cl’N9-C4 for purines. A positive torsion angle indicates a clockwise rotation of the farther bond with respect to the nearer one. (b) The three energetically favored conformations: gauche - for angles near -6O”, transfer 180” andgauche+ for +60”. Fig. 5. Correlation plot of main chain torsion angles (Yand y in all A-DNA fragments. Correlation coefficient is - 0.926 (2).

between the present RI2 values and the values obtained in previous studies shows an apparent decrease in several correlation coefficients when the sampling is increased. The first of these correlations is the [/a! +’ one. This structural relation was first noted by Shakked et al. [ 151. In the complete set of nine A-DNA structures, the observed value of R,, is lowered from - 0.755 in the study of Conner et al. [ 61 to - 0.517 (8). When the three cy angles with the tram conformations are omitted, this relation becomes slightly stronger ( -0.596( 7) ). It means that this correlation holds mainly for a! angles in the gauche- state, and vanishes for a truns conformation. In the five variants of the B-DNA dodecamer, d( CGCGAATTCGCG), which were studied in detail, the a! angle is frozen at the gauche - state, and therefore no correlation is observed with the preceding [ angle or any other angle. The c angle is however more variable in B-DNA than in A-DNA, and the strongest correlation in the backbone of B-DNA is that between E and c. The correlation between E andx torsion angles in A-DNA has been noted in the previous studies [ 1,6,15]. It has been attributed by Conner et al. [6] to a crankshaft motion involving the entire sugar ring. When calculated for all cur-

b

C Fig. 6. (a) Folded backbone conformation in A-DNA (AI). (b) Extended backbone conformation in A-DNA (An). (c) Projected views of various base-pair doublets in A-DNA crystal structures: Purine-purine (left), purine-pyrimidine (middle), pyrimidine-purine (right The views are perpendicular to the mean plane through the upper base.

).

rently available A-DNA structures, including all residues, this correlation becomes less significant, - 0.56 (1)) compared to - 0.71 from the previous study

tf31.

The e/j?+l correlation, like the C/X one, is only marginally significant when the calculation is carried out on all A-DNA duplexes (&= -0.571(9) ). In the B-DNA-dodecamer variants this correlation is meaningful. In A-DNA structures its significance is uncertain, although it is the second most significant correlation in Table 3. To summarize, the most important structural linkage in the sugar-phosphate backbone of A-DNA is the a/r one. It is observed in all A-DNA struc-

376 TABLE 3 Torsion angle correlations in A-DNA helices Angl

Ang2

P

+1

6 t I

4

X a+’ +1 B +1 Y ; X a+’

KS!”

o(R)

- 0.534 - 0.926 0.105 -0.370 0.171 0.325 -0.150 0.302 0.123 0.114 -0.051 0.211 - 0.006 - 0.044 - 0.003 0.291 -0.173 -0.239 0.101 - 0.023 -0.114 - 0.049 -0.323 0.494 0.102

0.007 0.002 0.009 0.009 0.010 0.011 0.005 0.008 0.012 0.012 0.013 0.013 0.009 0.012 0.009 0.008 0.009 0.010 0.005 0.009 0.005 0.013 0.013 0.011 0.011

Angl

Ang2

&s

o(R)

0.091 -0.199 0.225 -0.527 - 0.565 0.422 -0.571 -0.288 -0.113 - 0.053 0.158 -0.517 0.508 0.362 - 0.076 0.146 -0.115 -0.199 0.208 0.105 0.155 -0.145 - 0.032 0.178

0.013 0.011 0.013 0.009 0.011 0.007 0.009 0.008 0.011 0.612 0.014 0.008 0.010 0.008 0.012 0.012 0.013 0.011 0.013 0.012 0.014 0.015 0.015 0.015

“R,, is the two-variable correlation coefficent and a(R) is its estimated error. R,, is defined as: where S, is: nZiXf - (&Xi)’ and Sy is: n&Yf - (CiYi)“. R,,=n~iXiYi-C,Xi~iYi/(SxSy)“’ d(R) is calculated as follows: a’(R) =ZF=1 (aR/aXi)2a’(Xi) + (aR/aYi)‘ff’( Yi) where o(Xi) and a( Yi) are the estimated errors in the variables Xi and Yi (D. Babinovich, private communication). The use of a( R ) is more convenient than the use of statistical tables of significance. Here we calculated a(R) assuming a constant 1” error in each of the two concerned angles. A more realistic error is 10” and for such estimate all values of o(R) given in the following tables should be multiplied by 10. An alternative measure of the error in a particular torsion angle is the r.m.s. deviation from the mean.

tures determined to date. This correlatioin divides the A form into two conformational domains: a major domain (A,) with the gauche -,guuche + conformation for a! and y, and a minor conformational domain (An) where these angles adopt the trans,trans state. Analogously, in the B-DNA backbone the largest flexibility is observed for Eand c torsion angles which are strongly correlated. This linkage divides the B form into two conformational domains: Br ( trmqgauche - ) and Brr (gauche -, truns ).

The sugar-phosphate backbone conformation is, in general, less affected by the sequence of the bases than the base-stacking geometry discussed later. The major exception is the An conformation in A-DNA which has been observed so far in guanine residues at C-G linkages. Base stacking modes and local parameters An important aspect of local structure in DNA helices is the stacking geometry of successive base pairs along the double helix, which describes the degree of overlapping between the base pairs. Base stacking interactions are the driving force for helix formation and stability, and are now considered the main vehicle for inducing sequence-specific conformations. The relative arrangement of any two successive base pairs is fully defined by six degrees of freedom (three rotations and three translations). Of these, the parameters described in the following sections, two rotations and one translation, display significant variations within the various double helices, and are therefore used to characterize sequence-dependent structural features of the DNA double helix. The rotations are the helix twist, defined as the rotation from one base pair to the next about the helix axis, and the roll angle (OR) which describes the amount by which two adjacent base pairs open toward the minor or major groove [ 201. The relative displacement of two successive base pairs about their long axes, called slide (Fig. l),is a quantitative measure for the overlapping of two adjacent base pairs which is dependent on their distance from the helix axis [ 2,4,21]. The calculation of the slide parameter is independent of the position of the helix axis and hence can be used to estimate the extent of A versus Btype base-stacking geometry. Slide The slide parameter is derived from the coordinates of C6 and C8 atoms on two adjacent base pairs, and therefore its value is slightly sequence dependent (Table 4). As the C6 atom is in a five-membered ring and the C8 atom is in a six-membered ring, their positions are not equivalent, and hence the slide values calculated for a uniform model differ for the three possible base-pair doublets (purine-purine or R-R, 1.3 A; purine-pyrimidine or R-Y, 1.1 A; pyrimidine-purine or Y-R, 1.5 A). Nevertheless, signficant deviations from these values are observed in the single-crystal A-DNA structures. The C-G step in all A-DNA structures, exhibits the highest value for this parameter (Fig. 7, Table 4): e.g. 2.0 in d(CCCCGGGG) and 2.2 A in d( GGGCGCCC). The average slide values for these structures is around 1.6 A. On the other hand, G-C steps present in several of the A-DNA structures display the smallest slide values within each duplex: e.g. 0.7 A in d(GGGGCCCC ) and 1.0 A in d( GGCCGGCC). An extreme case is the G-C

378 TABLE 4 Average local helix parameters of identical base-pair steps”

G-G G-C G-T C-G T-A A-DNA

Helix twist (“)

Rise (A)

Roll (“)

Slide (8)

33.0(3.8) 32.0 (2.9) 33.4(3.1) 33.0(1.5) 34.3(O) 33.3(1.2) 26.9(6.2) 24.1(0.8) 29.3 (0.8) 29.0(0.9) 32.7

2.9(0.3)

5.8(3.4) 5.9(3.3) 5.8 (3.8) 9.0(3.8) 2.5(1.6) 2.2( 1.2) 8.4(4.4) 4.0(2.9) 15.8(3.9) 13.0(1.5) 10.3,13.8 or 12.1b

1.6(0.4) 1.7(0.3) l.O(O.5) l.O(O.3) 1.3(0.2) 1.4(0.2) l.B(O.3) 1.9(0.2) 1.4(0.1) 1.4(0.1) 1.5, 1.1 or 1.3b

3.0(0.2) 3.0(0.4) 3.2(0.1) 2.7(0.2) 2.8 (0.2) 3.1(0.6) 3.4(0.1) 3.0(0.2) 3.1(0.1) 2.56

“The figures in the first row of each base-pair step are the averaged values for all published singlecrystal A-DNA structures. The values in the second line are averaged only over the A-DNA etructures determined at room temperature (excluding the hybrid decamer, d(‘CCGG), and d( GGCCGGCC) ( - 8°C) ). bThe values are for C-G, G-C, and G-G base-pair steps derived from fiber-data coordinates.

2.5 2.0 s&1.5 1.0 0.5

0.01

12

3

4

5

6

7

vc-C-C-C-G-G-G-G DC-G-G-C-G-C-C-C OG-G-G-G-C-C-C-C OG-G-T-A-T-A-C-C

Fig. 7. Slide between successive base pairs as a function of sequence in four A-DNA structures. The values for duplexes not incorporating a crystallographic two-fold axis (space group I%,) were obtained by averaging equivalent steps (i.e. 1 and 7,2 and 6 etc.).

step of d(‘CCGG)/d(‘CCGG) with a slide value of 0.1 A. However, this situation probably arises from the absence of linking phosphates at the central step, and therefore does not represent a true A-type value. Slide values of G-

G steps occupy the gap between those of G-C steps at the lower end of the scale and those of C-G at the higher end. Helix twist Another parameter that adopts distinct values in C-G doublets is the helix twist (Table 4, Fig. 8). The helix twist angles of C-G steps manifest always the smallest values; e.g. 25’ in d (CCCCGGGG ) ,24’ in d (GGCCGGCC ) and d(GGGCGCCC).Th e average values for this parameter in the three structures are 34”) 33’) and 32’ respectively. Extremely low values for this step (16,18” ) were observed in d(GGCCGGCCCC) ( -S°C), and in d(GGm5CCGGCC). The small helix twist angles are accompanied by large ones in the flanking steps leading to an alternating pattern of helix twist values. Thus, a C-G step dominates the helix twist pattern of the whole duplex, leading to a similar pattern for all structures embedding such a step, even though the flanking base pairs are different (Fig. 8). The helix twist of G-C steps is not distinctive as that of C-G steps. It adopts values which are close to the average ones in d(GGGCGCCC) and d(GGCCGGCC) (35”, 34”) and somewhat lower (31”) in d(GGGGCCCC). As shown by Fig. 8, the helix twist of G-G sites spans a similar range of values. Both d (GGGGCCCC ) and d (CCCCGGGG ) incorporate identical homopolymer fragments but differ in their polarity (G-C versus C-G). The helix twist patterns of the two are very different, and demonstrate the conceivable influence of a single step on neighboring steps (Fig. 8). The small C-G twist 40(,,,,,,,, 38 3634 -

m

32302826ti

24 22

’ ’ 1234567

’

’

’

’

’

VC-C-C-C-G-G-G-G q

C-G-G-C-G-C-C-C

OG-G-G-G-C-C-C-C OC-G-T-A-T-A-C-C

Fig 8. Helix twist angles as a function of the sequence in four A-DNA structures. The values for duplexes in P6, were obtained as for Fig. ‘7.

angle at the center of d(CCCCGGGG) gives rise to large twist values at the adjacent steps, which are damped toward the helix ends. On the other hand, the twist pattern in d(GGGGCCCC) is rather monotonous as a result of a central G-C step adopting a twist angle comparable to that of homopolymer steps. Roll angle The roll angle is calculated from the average planes through successive base pairs. From the fiber-derived values (Table 4) we see that the roll angle is another “sequence-dependent” parameter. The best-plane calculation is carried out using all the ring atoms of the base pair. As the bases within a pair are propeller twisted, the mean plane through a base pair is biased towards the large purine base, thus creating the observed dependence on the sequence. Other atoms could have been used in the calculations in order to diminish such discrepancies. However, the present calculations have been carried out in the above manner for an easier comparison with previous studies of DNA crystal structures [ 1,2,4]. The roll angle is also a highly variable parameter (Fig. 9). However, it appears to be softer and less sequence-specific than the other parameters determining local structure of DNA helices. This is reflected in the high standard deviations calculated for similar steps (Table 4). The roll angle appears to be influenced by external factors such as temperature, hydration, and crystal forces. For example, in structures determined at relatively low temperatures,

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VC-C-C-C-G-G-G-G UC-G-G-C-G-C-C-C oG-G-G-G-C-C-C-C oG-G-T-A-T-A-C-C Fig. 9. Roll angles between adjacent base pairs as a function of the sequence in four A-DNA structures. The values for duplexes in ES, were obtained as for Fig. 7.

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C-G steps exhibit high roll angles and G-C steps low values whereas in the room-temperature structures the situation is reversed. The roll patterns in d (GGGCGCCC ) and in d (GGGGCCCC ) which differ only in the order of the two central base pairs are very different (Fig. 9). In d (GGGCGCCC ) the lowest roll angle is at the central C-G step and increases monotonously toward the edges; in d(GGGGCCCC) the central G-C step has the highest value, which decreases thereafter toward the helical ends. The different features of the terminal GGG regions of the two structures could have been induced by end effects, or crystal forces which differ for the two. However, it may well be that here again we observe the effect that a single base-pair step can have on its neighboring steps, as already demonstrated for the helix twist angle. Base stacking energy Base-stacking energy calculations may be used for a better understanding of the observed variability in the stacking modes of DNA double helices [ 4,221. The van der Waals’ stacking energies calculated for the structures of d(CCCCGGGG), d(GGGCGCCC), d(GGTATACC) andd(GGGGCCCC) are shown in Fig. 10. For each base-pair doublet the total energy and its intra- and interstrand components are given. The energy functions used for the calcula0

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tions were described previously [ 221. The main conclusion based on such calculations is that local changes in the various helical parameters decrease the overall stacking energy, and at the same time damp down the energy differences between helical steps. The total van der Waals’ stacking energy is therefore equipartitioned along the double helix. The relative contributions to the total stacking energy, from interactions within each strand and from those between strands, are different for each base-pair doublet, and therefore have to be complementary. The intrastrand stacking energy at pyrimidine-purine steps (e.g. C-G) of A-DNA structures is significantly higher than that at other steps, because as mentioned above, intrastrand stacking of such doublets is poorer than that of other doublets. On the other hand, the interstrand stacking energy is significantly lower at C-G doublet than for other doublets. In d (CCCCGGGG) and d( GGGCGCCC) the two contributions for C-G are almost identical. Their interstrand components are the lowest in this series, as a consequence of the considerable stacking of the two guanines from opposite strands. This stacking geometry is achieved by the high slide and low propeller twist adapted by the C-G doublet. For G-C steps, the intrastrand energy is always lower than that of other steps demonstrating that the predominant stabilization of these doublets comes from intrastrand interactions. As expected, the interstrand contribution to the overall stacking energy in these doublets is relatively small. G-G steps in A-DNA structures also display similar patterns of stacking energy. The major contribution is from intrastrand interactions where G-G contribution is about - 10 kcal mol-’ and C-C is -5 kcal mol-l. However, a significant contribution ( - 5 kcal mol-’ ) comes also from intrastrand stacking between guanine and cytosine bases of opposite strands. It demonstrates that in homopolymer steps the cystosines do not wrap passively around the double helix, but rather contribute significantly to the stability of the double helix via both intrastrand and interstrand interactions. Although G-G steps display a wide range of stacking arrangements, their stacking energies are almost identical. These findings show that the invariant quantity characterizing base-pair doublets in right-handed double helices is the van der Waals stacking energy. The total stacking energy is similar for all helical steps, leading to the equipartitioning of the energy along each double helix and between duplexes. In contrast to the total energy, the components of this energy are sequence specific, and therefore are invariant only within any doublet type. This invariance is brought about by local modulations in the relative orientation and translation of adjacent base pairs. Mechanism of inducing optimized base stacking arrangements In the preceding section we have seen that intrastrand and interstrand basestacking interactions of C-G steps are of comparable importance, whereas in

G-C steps intrastrand stacking contributes almost exclusively toward the overall stacking energy of this step. As in many other aspects of local structure in DNA helices, G-G steps are found between the two extreme situations. The interstrand contribution to the total energy of the homopolymer sites is about one third of the intrastrand one. How do different steps achieve optimized stacking arrangements? The mechanism for an optimized stacking geometry of successive base pairs has some features which are common to all doublets of the same type, regardless of the flanking base pairs, whereas other features are dependent on the identity of the neighbors. These features are discussed for each of the three major basepair doublets: C-G, G-C and G-G. The available information on the other steps is small and hence a general pattern cannot be derived yet. C-G steps All known C-G steps in A-DNA crystal structures (excluding the RNADNA structure, the odd-one-out structure in many respects) display the highest slide values and the lowest helix twist angles (Figs. 7,8 and Table 4). The relative sliding of guanines in C-G steps is the main tool for improving their interstrand base stacking, at the expense of intrastrand stacking. Closing down the twist angle increases mainly the intrastrand stacking of the two concerned base pairs. The extended backbone conformation, observed for the guanine residues of these steps, appears to facilitate these two motions, or rather one motion which is a combination of slide and twist. Therefore, in a two-dimensional plot of slide versus helix twist (Fig. 11)) the C-G steps cluster at a region of very high slide and very low twist. The sliding/twisting motion results in a large intrastrand P-P distance (7 A) between the guanine phosphate and the next one at the 3’-side. G-C steps The only local parameter displaying typical values for G-C steps is the slide. G-C steps are characterized by the smallest slide values in each helix, indicating a better intrastrand stacking compared to other steps. Unlike C-G steps, the stacking geometry of G-C is sensitive to the identity of neighboring steps. For example, in d (GGGCGCCC) the G-C step is adjacent to the dominant CG step, whereas in d( GGCCGGCC) and d( GGGGCCCC ) the same steps are surrounded by homopolymer tracts. A relatively large twist in the first structure (35 ’ ) is induced by the low twist of the adjacent C-G step, so as to maintain an overall value of 32’. In the other two structures G-C steps adopt values (33,31 o ) which are similar to the average ones. The tendency of G-C steps to adopt low slide values, which improve their intrastrand overlap, is disturbed when adjacent to the dominant C-G step. The G-C steps flanking the central C-G step in d(GGGCGCCC) are characterized by the largest slide values (1.3

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G-G steps As mentioned above, the stacking pattern of G-G doublets is highly variable. These steps display a broad spectrum of twist, roll, and slide values. The specific values are highly influenced by neighboring helical steps. For example, G-G sites adjacent to C-G ones display high twist, low roll and medium slide, whereas the ones at the vicinity of G-C sites exhibit medium values of the three parameters, similar to the ones shown by homopolymer tracts.

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Clustering and correlation patterns in local parameters of A- and B-DNA

It is instructive to look at the distribution of several parameters characterizing the local geometries of base-pair doublets in A and B-DNA. One looks for either two-variable correlations as we have already done for torsion angles, or for clustering patterns, such as the slide-twist clustering of C-G steps observed in A-DNA. Plots of two-variable combinations of helical parameters for A and B-DNA helices are shown in Figs. 11-16. The values for B-DNA were taken from two variants of the B-dodecamer which were determined most accurately: the native structure d( CGCGAATTCGCG) determined at 2O”C, manifesting an 18” axial bend, and the 9-Br analogue from high MPD deterI

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mined at 7’ C, with a negligible bend (Dickerson et al. [ 21). The values for ADNA structures were taken from: d( GGTATACC ) , d (GGGGCCCC ), d(‘CCGG), d(GGCCGGCC) determined at 15 and -8”C, d(CCCCGGGG), d(GGGCGCCC), d(GGGTACCC) and r(GCG)d(TATACGC) (references are given in Table 1) . There are several ways of examining these plots. First, one can learn about the clustering/correlation/random patterns of various base-pair doublets within either A or B-DNA structures. Secondly, one can compare the patterns in A versus B-DNA for a specific step. Thirdly, one can look for the parameters or combination of parameters most distinguishing an A helix from a B one, as well as for the overlapping regions of A and B-DNA which are relevant to the A to B transition. These points and other aspects emerging from these diagrams, are discussed below. The helix twist and rise per base pair values in A and B-DNA show large common regions (Figs. 11 and 13). The roll angles display a common region consisting of nearly half of the steps in A and B-DNA (Fig. 12). The slide is

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the only local parameter where no overlap is observed between values of A and B-DNA for all types of base-pair steps (Figs. 11-13). In B-DNA the maximum slide is 0.6 A whereas in A-DNA this is the smallest value observed so far. Therefore, in all three diagrams having slide as one of the parameters two distinct regions are occupied by A and B-DNA (Figs. 11-13). Calladine and Drew [21] have discussed the two-dimensional plot of roll versus slide in relation to the bistability of Y-R (pyrimidine-purine) steps. They have suggested that the A to B transition is carried out by a change in these two parameters only. However, now that we have observed low roll values for C-G and G-C steps in A-DNA helices and that the region of high roll is occupied by G-G and G-C steps as well, the slide-roll pattern has to be modified. The slide-roll plot is still the one showing maximal separation between A-DNA and B-DNA zones. This is a consequence of the separation observed for each pa-

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rameter by itself. However, for several Y-R steps, a change in the slide parameter only appears to be sufficient for a B-A transition whereas for other Y-R points, the slide-roll pathway may still be valid. The transition from B to A can be also described by a slide-twist pathway for certain Y-R points (Fig. 11). Changes in only these two parameters that is, increase in slide and a decrease in twist, will bring the Y-R points of the BDNA region to those of the A-DNA region. Likewise, the B to A transition can be described by a slide-rise pathway for some points (Fig. 13); the extreme case being from C-G steps of B-DNA (high rise and low slide ) to C-G steps of the RNA-DNA hybrid structure (low rise and high slide). These data confirm that Y-R steps are bistable, and have unique characteristics. However, one cannot conclude from the above that the B-A transition can be described by two parameters only. The transformation from one form to the other, probably

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utilizes changes in all helical parameters. Hence, the only conclusion we can make at present is to single out the slide as the local helical parameter differentiating between the two right-handed forms, and the most important one for mediating the A to B transition. All other helical parameters are probably of secondary importance in that respect, and are utilized selectively in different cases according to the identity of the base-pair doublet and the flanking ones. Calladine and Drew [21] have also suggested that the bistability of any step can be transmitted to the adjoining steps by the sliding motion only. Looking at the A-DNA regions we note that R-Y (purine-pyrimidine) and Y-R (pyrimidine-purine) steps are characterized by clustering or correlation patterns for certain two-variable combinations whereas R-R (homopolymer ) steps do not show any unique patterns. The most distinct division into the above two categories is observed in the slide-roll plot, with a clustered R-Y region and modestly correlated Y-R region (Fig. 12). The R-Y region is clustered at low slide and low to medium roll, and the Y-R region is stretched above it at high slide values. The slide and roll values of Y-R doublets in A-DNA are anticorrelated with a coefficient of - 0.8. T-A steps are found at medium slide and high roll region, and C-G steps at the high slide and low-to-medium-roll regions. Another division into R-Y and Y-R regions of A-DNA can be seen in the slide-twist diagram (Fig. 11). The slide-twist variations for all Y-R steps are

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slightly anticorrelated with a coefficient of -0.6 whereas for C-G steps only the relation becomes stronger with R12= -0.8. Within the Y-R region, C-G steps are clustered at high slide and very low twist, whereas T-A steps are clustered at medium slide and low-twist domain. R-Y steps are located at medium twist and low-to-medium-slide domain. In the diagram showing the variation of twist versus rise values in A-DNA (Fig. 14) the majority of Y-R steps are located at low twist and medium to high rise values, except for two C-G steps from the hybrid structure which exhibit very low rise (2.3 A) and relatively high twist values (35’ ). The variations in the two parameters are highly anticorrelated, with RI2 of -0.9 which rises to - 0.95 for C-G steps only, and even further to - 0.99 when the hybrid C-G steps are omitted. Within the Y-R region C-G steps cluster at the central domain, and T-A cluster at the right-handed one, as observed also in the slidetwist plot. Most R-Y steps are located at medium twist and medium rise values. In all these plots the R-Y region is much more compact than the Y-R region. The typical behavior of Y-R versus R-Y steps is not shown in the other plots (Figs. l&16). Based on the above data we conclude that some features of local structure in DNA helices are of a more general character than others, and hold for most members of Y-R or R-Y base-pair doublets. In several two-variable plots the separation between Y-R and R-Y regions is very clear. The stacking geometry of R-R doublets is context dependent, that is, they are affected by base pairs adjoining them on either side. In A-DNA the slide parameter is highly sequence dependent. It is also the best measure for characterizing A versus B-type structures. ACKNOWLEDGEMENTS

We thank our colleagues D. Rabinovich and M. Eistenstein for help and discussions. We thank A. Wang and A. Rich for fruitful collaboration and for supplying some of their unpublished data. We also thank the Minerva Foundation, Munich, Germany, the United-States/Israel Binational Science Founcation (BSF) and the Israel Cancer Association for financial support. Z.S. holds the Helena Rubinstein professorial chair in structural biology.

REFERENCES 1

2

Z. Shakked and 0. Kennard. In A. McPherson and F. Jurnak, (Eds.), Structure of Biological Macromolecules and Assemblies, Vol. II: Nucleic Acids and Interactive Proteins, Wiley, New York, 1985, pp. l-36. R.E. Dickerson, M.L. Kopka and P. Pjura. In: A. McPherson and F. Jurnak, (Eds.), Structures of Biological Macromolecules and Assemblies. Vol. II: Nucleic Acids and Interactive Proteins, Wiley, New York, 1985, pp. 38-126.

391 A.H.-J. Wang and A. Rich. In: A. McPherson and F. Jurnak, (Eds.), Structures of Biological Macromolecules and Assemblies. Vol. II: Nucleic Acids and Interactive Proteins, Wiley, New York, 1985, pp. 128-170. 4 Z. Shakked and D. Rabinovich, Prog. Biophys. Mol. Biol., 47 (1986) 159. 5 0. Kennard in D. Lilley and F. Eckstein (Eds.), Trends in Nucleic Acids Research, Springer Verlag, Heidelberg, 1986. 6 B.N. Conner, C. Yoon, J.L. Dickerson and R.E. Dickerson, J. Mol. Biol., 174 (1984) 663. I.H. Hall, L.C. Puigjaner, J.K. Walker and M. Wang, Cold 7 S. Arnott, R. Chandrasekaran, Spring Harbour Symp., Quantum Biology, 47 (1982) 53. 8 T.E. Haran, Z. Shakked, A.H.-J. Wang and A. Rich, J. Biomol. Struct. Dynam., 5 (1987) 199. 9 D. Rabinovich, T.E. Haran, M. Eistenstein and Z. Shakked, J. Mol. Biol. 200 (1988) 151. 10 T.E. Haran, Z Shakked, A.H.-J. Wang and A. Rich (1988), in preparation. 11 CA. Frederick, D. Saal, G.A. van der Marel, J.H. van Boom, A.H.-J. Wang and A. Rich, Biopolymers, 26 (1987) 145. 12 C.A. Frederick, M. Teng, G.J. Quigley, A.H.-J. Wang, A. Rich, G.A. van der Marel, J.H. van Boom and Z. Shakked, Book of abstracts, 5th Conversation in Biomolecular Stereodynamics, R.H. Sarma (Ed.), 1987, Albany. 13 F.H.C. Crick and J.D. Watson, Proc. R. Sot. London Ser. A., 223 (1954) 80. 14 W.K. Olson, Nut. Acids Res., 10 (1982) 777. 15 Z. Shakked, D. Rabinovich, 0. Kennard, W.B.T. Cruse, S.A. Salisbury and M.A. Viswamitra, J. Mol. Biol., 166 (1983) 183. 16 M. McCall, T. Brown and 0. Kennard, J. Mol. Biol., 183 (1985) 385. 17 A.H.-J. Wang, S. Fujii, J.H. van Boom and A. Rich, Proc. Natl. Acad. Sci. U.S.A., 79 (1982) 3968. 18 M. Eisenstein, F. Frolow, Z. Shakked and D. Rabinovich, (1988) in preparation. 19 A.H.-J. Wang, S. Fujii, J.H. van Boom, G.A. van der Marel, S.A.A. van Boeckel and A. Rich, Nature, 299 (1982) 601. 20 A.V. Fratini, M.L. Kopka, H.R. Drew and R.E. Dickerson, J. Biol. Chem., 257 (1982) 14686. 21 C.R. Calladine and H.R. Drew, J. Mol. Biol., 178 (1984) 773. 22 T.E. Haran, Z. Berkovitch-Yellin and Z. Shakked, J. Biomol. Struct. Dynam., 2 (1984) 397. 23 S. Jain, G. Zon and M. Sundaralingam, J. Mol. Biol., 197 (1987) 141. 3