Adenosine-Proflavine Complex: a Self-Paired Parallel-Chain

Home , Proflavine

Proc. Natl. Acad. Sci. USA Vol. 77, No. 4, pp. 1852-1856, April 1980 Biochemistry X-ray structure of a cytidylyl-3',5'-adenosine-proflavine complex: A self-paired parallel-chain double helical dimer with an intercalated acridine dye* (non-self-complementary pairing of adenine-adenine and protonated cytosine-cytosine/intercalation dynamics/ sugar-phosphate backbone stereochemistry/frameshift mutagenesis) E. WESTHOF AND M. SUNDARALINGAM Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 Communicated by David R. Davies, December 31, 1979

ABSTRACT The non-self-complementary dinucleoside parallel-chain double helical dimer of cytidylyl-3',5'-adenosine monophosphate cytidylyl-3',5'-adenosine (CpA) forms a base- (CpA). The dinucleoside monophosphate CpA was chosen not paired parallel-chain dimer with an intercalated proflavine.The dimer complex possesses a right-handed helical twist. The dimer only because it constitutes the end portion of the invariant CCA helix has an irregular girth with a neutral adenine-adenine (A-A) terminus of all tRNAs, but also because it cannot form self- pair, hydrogen-bonded through the N6 and N7 sites (C1'... C1' complementary pairs. separation of 10.97 A), and a triply hydrogen-bonded protonated cytosine-cytosine (C-C) pair with a proton shared between the METHODS base N3 sites (C1'... C1' separation of 9.59 A). The torsion an- EXPERIMENTAL gles of the sugar-phosphate backbone are within their most Single crystals of a 1:1 complex of CpA and proflavine were preferred ranges and the sugar puckering sequence (5' - 3') is grown from an equimolar aqueous solution of proflavine he- C3'-endo, C2'-endo. There is also a second proflavine molecule have space sandwiched between CpA dimers on the 21-axis. Both proflav- misulfate and the sodium salt of CpA. The crystals ines are necessarily disordered, being on dyad axis, and this group P42212 with unit cell parameters a = b = 19.38 (1) A and suggests possible insights into the dynamics of intercalation of c = 27.10 (1) A. Intensity data were collected at 100C with an planar drugs. This structure shows that intercalation of planar Enraf-Nonius (CAD-4) diffractometer. Of the 5214 indepen- drugs in nucleic acids may not be restricted to antiparallel dent reflections measured to a resolution of 0.8 A, 2574 had complementary Watson-Crick pairing regions and provides intensities of at least three standard deviations. The structure additional mechanisms for acridine mutagenesis. was solved by a combination of Patterson techniques, direct The genetic molecules DNA and RNA are targets for various methods (MULTAN) (15, 16), and Fourier synthesis. The chemical agents that possess carcinogenic and mutagenic structure was refined initially by block diagonal least-squares properties. The molecular modes of interactions of these agents analysis with isotropic temperature factors. The discrepancy with the nucleic acid and the effects they produce on the mo- index R (where R = 1IFO|- IFCII/IFo| and Foand Fc are lecular geometry and conformations of DNA and RNA have the observed and calculated structure amplitudes, respectively) been a subject of considerable interest (1-4). X-ray diffraction was 15% for 2460 reflections with the full CpA molecule, the studies of DNA fibers (2, 5) and of co-crystals of self-comple- proflavine molecule, and 10 water molecules (two full sites and mentary dinucleoside monophosphate (6, 7) with the dye eight partially occupied sites). The introduction of anisotropic proflavine have provided important insights on the stereo- temperature factors for the CpA molecule together with the chemistry of intercalation of proflavine into nucleic acids. use of counting statistics for the weighting scheme decreased Because the known single-crystal complexes, in which the dye R to 14%. Full matrix least-squares refinement of the proflavine is intercalated between the base pairs, involve self-comple- molecule and the introduction of five additional partially oc- mentary chains, it was of interest to investigate whether similar cupied water sites decreased R to 12%. The details of the crys- complexes can be formed between dyes and non-self-comple- tallographic refinement will be published elsewhere. mentary nucleic acid fragments. Also, it has been demonstrated that proflavine causes a direct inhibition of protein synthesis RESULTS AND DISCUSSION (8, 9) and that it impairs the enzymic aminoacylation of tRNA CpA Forms a Parallel Dimer with a Protonated C-C Pair (10). Binding studies led to the conclusion that proflavine in- and a Neutral A-A Pair. The asymmetric unit of the crystal hibition of protein synthesis results from strong binding of consists of one CpA molecule (Fig. 1) and one proflavine mol- proflavine to tRNA (11, 12). The evidence for intercalation of ecule. The proflavine of the asymmetric unit consists of two proflavine in tRNA was, however, indirect (11, 12). The binding proflavine molecules lying on the dyad axis at different posi- of proflavine to yeast tRNAPhe was studied by x-ray crystal- tions, each contributing half-weight. The CpA molecules related lography after diffusing proflavine into pregrown crystals of by a dyad coincident with the 42- or 22- axis of the crystal are yeast tRNAPhe (13). Intercalation of proflavine in helical stems self-paired to form a right-handed parallel-chain dimer (Fig. was not found; instead, the binding sites involve electrostatic 2). The two-fold symmetry-related cytosine bases are paired and hydrogen bonding interactions with the polynucleotide with three hydrogen bonds: two between the exocyclic atoms sugar-phosphate backbone (14, t). 02 and N4 and one between the ring nitrogens N3. This pairing Here, we report the crystal and molecular structure of an intercalated proflavine in a novel self-paired right-handed Abbreviations: CpA, cytidylyl-3',5'-adenosine; Tm, amplitude of puckering. The publication costs of this article were defrayed in part by page * Presented at the American Crystallographic Association Summer charge payment. This article must therefore be hereby marked "ad- Meeting, Boston, MA, August 1979, Abstr. S1. vertisement" in accordance with 18 U. S. C. §1734 solely to indicate t Liebman, M. N., Rubin, J. & Sundaralingam, M. (1977) American this fact. Crystallographic Association Meeting Abstracts, East Lansing, MI. 1852 Downloaded by guest on September 25, 2021 Biochemistry: Westhof and Sundaralingam Proc. Natl. Acad. Sci. USA 77 (1980) 1853 observed in the crystal structure of cytosine-5-acetic acid (18) (see also below). The dyad-related adenine bases are paired through the N6 and N7 sites (distance 2.935 + 0.015 A). This pairing scheme between adenine bases has been proposed for acid poly(A) (19) and has been observed in several Ni-protonated adenine nucleotide systems (20-23). From the value of the internal angle at N1 and from considerations of charge neutrality (see below), it appears that the adenine bases are neutral. Intercalated and Sandwiched Proflavines. One of the proflavine molecules is intercalated between the C-C and A-A self pairs, whereas the other proflavine is sandwiched between the terminal A-A and C-C pairs of 21-related helical fragments (Fig. 2). Because the proflavine molecules do not have a dyad axis normal to their ring planes, they cannot obey the two-fold symmetry and are necessarily disordered. The intercalated proflavine is two-fold disordered with the dyad axis passing off the center of the middle ring (Fig. 3 Upper). In the case of the N7 sandwiched proflavine, the dyad axis passes through the center of the middle ring, essentially placing the exocyclic amino groups at half-occupancy at four positions (Fig. 3 Lower). Charge Balance. Because no sulfate anion was located in the Fourier maps, the hemiprotonation of the cytosine base implies that the proflavine molecule is hemiprotonated and that the adenine base is neutral, with the charge balance accomplished by the negative charge on the anionic oxygens of the phos- N3~ phodiester group (or C'/2+p-A + proflavinel/2+). Thus, a CpA FIG. 1. An ORTEP (17) drawing of CpA. The atoms are repre- dimer has one net negative charge and the two proflavine sented by their thermal ellipsoids which are scaled to a 20% proba- molecules have together one net positive charge. The disorder bility level. The atom numbering and the notations for the torsion of the proflavines makes it difficult to give a definite identifi- angles are indicated. The gauche+ (g+) domain corresponds to torsion cation of the protonation scheme: either the sandwiched angle values centered on +60°; the trans (t), on 1800; and thegauche proflavine protonated with the intercalated proflavine neutral, (g-), on -60°. or vtice versa, or there was statistical disorder in which the proton was shared between the sandwiched and intercalated scheme strongly suggests that the cytosine bases are hemipro- proflavines. In any case, on an average, half the proflavine tonated, a proton being shared between the N3 nitrogens of molecules are neutral. The loss of one proton by the proflavine

dyad-related molecules (N3 ... N3 distance is 2.876 0.015 molecules to the cytosine pair is unexpected in consideration A). A similar pairing scheme between cytosine bases has been of the pK values (24, 25) of proflavine (quaternary nitrogen: 9.7) and of cytidine-5'-phosphate (N3:4.5) and of the pH value of the crystallization solution (n 6.0). However, this apparent anomaly may be explained by the fact that the protonation of the C-C pair leads to a very stable structure, which shifts upward the pK of the cytosine base. Indeed, alkaline titration studies of poly(dC) (26) and poly(rC) (27-29) have shown that the pH of the melting transition (pHm) is shifted upwards from the pK of cytosine. It has further been shown that the magni- tude of the shift (pHm minus pK) is proportional to the stability of the base-paired helix-i.e., the melting temperature, Tm (26, 27). Geometry of Dimer Helix. The planes of the cytosine and adenine bases of the CpA chain make a dihedral angle of 3.5°. The C-C and A-A base pairs are separated by a distance of about 6.79 : 0.02 A. The angle between the vectors connecting the C1' atoms of hydrogen-bonded self-pairs is about 40°, which is 50 smaller than the value of 450 proposed for parallel-chain double helical poly(A) (19) ("winding") and 100 greater than the value of 300 for a proposed double helical structure of poly(C) ("unwinding") (30). The corresponding winding angles for antiparallel double helical B-DNA and RNA-11 are respectively 360 and 330 (31). The C1'-Cl' distances for the two FIG. 2. Drawing ofself-paired parallel dimer ofCpA showing the pairs are unequal and the value for the A-A pair (10.97 + 0.02 three-hydrogen-bonded protonated C-C pair and the two-hydro- A) is significantly higher than the value for the C-C pair (9.59 gen-bonded neutral A-A pair. The intercalated proflavine (center) ± 0.02 A); thus, the dimer helix is irregular. It may be noted and the sandwiched proflavines (above and below) are shown. For that, the clarity, only one disordered site of each proflavine is shown. The hy- because dyad is parallel to the helical axis and not drogen bond between the amino group of the intercalated proflavine perpendicular to it as in Watson-Crick double helices, the and one anionic phosphate oxygen is shown. glycosyl bonds of the self-pairs are in the trans configuration Downloaded by guest on September 25, 2021 1854 Biochemistry: Westhof and Sundaralingam Proc. Natl. Acad. Sci. USA 77 (1980)

C1,

2HN

C 1,

Cl,

C 1 'N.o Cl'~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~~l1

FIG. 3. (Upper) View down the dyad axis illustrating the stacking of the C-C pair (Left) and the A-A pair (Right) with the two sites occupied by the disordered intercalated proflavines. (Lower) View down the dyad axis illustrating the stacking of the C-C pair (Left) and the A-A pair (Right) with two sites occupied by the disordered sandwiched proflavines. in contrast to the cis configuration of antiparallel Watson-Crick The stacking between the intercalated proflavine molecule helices. and the self-pairs of cytosine and adenine bases is such that the Hydrogen Bonding, Solvent Interactions, and Stacking of overlap of-the proflavine with the protonated cytosine self-pairs Drug with Bases. The hydrogen bonding network is intricate is more extensive than with the neutral adenine self-pairs (Fig. and involves several disordered water molecules. In each of its 3 Upper). In fact, there is hardly any overlap of the proflavine two occupied sites, the intercalated proflavine interacts with with the adenine rings. The nonintercalated proflavine is only one backbone chain through hydrogen bonding between sandwiched between the cytosine self-pairs of one duplex and one of the exocyclic amino groups and an anionic phosphate the adenine self-pairs of a symmetry (42)-related duplex with oxygen. The ring nitrogen of the intercalated proflavine is their long axis roughly parallel to the C6-C6 vector of cytosine hydrogen bonded to a water molecule. This water molecule lies pairs and to the C4-C4 vector of adenine pairs (Fig. 3 Lower). about 3.4 A over each adenine ring resulting from the two-fold Unlike the intercalated proflavine, this proflavine overlaps disorder of the proflavine molecule. Besides the base sites that extensively with both the C-C and A-A base pairs. are engaged in base pairing, the remaining potential hydrogen Conformation of Sugar-Phosphate Backbone. The values bonding sites in the bases are involved in interaction with sol- of the various torsion angles in the sugar-phosphate backbone vent molecules or neighboring CpA molecules. The adenine are given in Table 1. The values observed here for the extended exocyclic amino group N6 is linked through a water molecule parallel helical fragment are very close to those reported for to the phosphate oxygen involved in hydrogen bonding with extended antiparallel helical fragments (4). These values con- the amino group of the intercalated proflavine. A water mol- form also to the principles embodied in the "rigid" nucleotide ecule bridges the carbonyl oxygen 02 of the cytosine base and theory (34), for instance: (i) the internucleotide phosphodiester the ribose 2'-hydroxyl oxygen atom of the same nucleotide. The torsion angles (w, w') adopt the characteristic helical gauche-, 2'-hydroxyl oxygen atom of the adenosine residue is hydrogen gauche- (g-, g-) conformation; (ii) the angles ) and c' are in bonded to an amino group of a neighboring sandwiched the usual trans (t) domain, though the angle 02 is on the outer proflavine, whereas the 2'-hydroxyl oxygen of the cytidine limit of the preferred domain; ('ii) the torsion angles L' about makes a similar hydrogen bond with the intercalated proflavine C4'-C5' of the cytosine and adenine riboses are both in the of an adjacent duplex. gauche + (g+) region. The puckering sequence 5' 3' is Downloaded by guest on September 25, 2021 Biochemistry: Westhof and Sundaralingam Proc. Natl. Acad. Sci. USA 77 (1980) 1855

Table 1. Sugar-phosphate-sugar backbone torsion angles in CpA-PF and related structures Xi iIl Pucker 5' 0Vk' (k202 112 Pucker 3' X2 rCpA-PF 12 56 C3'-endo 208 284 293 223 62 C2'-endo 84 rI5CpG-EtBr (32) 29 51 C3'-endo 226 281 286 210 72 C2'-endo 101 24 90 C3'-endo 225 291 291 224 55 C2'-endo 109 rCpG-PF (7) 17 C3'-endo 201 290 289 231 52 C3'-endo 85 dApTpApT (33) 0 68 C3'-endo 213 294 293 176 68 C2'-endo 70 0 64 C3'-endo 212 284 302 171 64 C2'-endo 70 PF, proflavine; EtBr, ethidium bromide; r, ribose; d, deoxyribose; CpG, cytidylyl-3',5-guanosine; I5CpG, 5-iodo-CpG; ApT, adenylyl-3',5'- thymidine.

C3'-endo-C2'-endo-i.e., the 5'-residue has a C3'-endo is 350 + 1°) than the C2'-endo ribose (rm is 430 + 10). These puckering and the 3'-residue has a C2'-endo puckering. The values deviate +40 from the average value observed in ribosides glycosyl torsion angles X of both nucleotide residues are in the (Tm = 390) (36). In the published structures of self-comple- preferred anti domain and are correlated with the puckering mentary dimers with or without drug, the amplitudes of mode; the cytidine residue in the C3'-endo pucker has a low puckering are similar for the different riboses of a chain. Thus, X value, whereas the adenosine residue in the C2'-endo pucker it appears that, besides the phase angle of pseudorotation, has a high X value. variations in the amplitude of puckering (37) might be im- It is interesting that, despite the formation of an intercalated portant in governing the stereochemistry and dynamic func- parallel complex, the polynucleotide chain displays the alter- tions of nucleic acids. nating puckering sequence that is usually observed in intercalated antiparallel dimer complexes not involving proflavine (4). CONCLUDING REMARKS However, in the antiparallel dimer structures with proflavine The crystal structure presented in this paper shows that the as an intercalator (6, 7), both riboses have a C3'-endo pucker. intercalation of planar drugs between base pairs of dimers is The alternating puckering sequence observed in the CpA not restricted to antiparallel chains with complementary complex may be related to the fact that each of the disordered Watson-Crick base pairing. It is conceivable that the proflavine intercalated proflavines is hydrogen-bonded to only one of the has induced this novel self-paired right-handed parallel dimer phosphate backbones and not to both phosphate backbones si- of CpA by protonating the C-C pair. Thus, it would appear that multaneously as in the antiparallel r-5-iodocytidylyl-3',5'- proflavine can stabilize self-pairs of neutral adenine bases and guanosine and cytidylyl-3',5'-guanosine (7) proflavine com- induce self-pairs of protonated cytosine bases between parallel plexes. Therefore, hydrogen bonding of the proflavine to one chains of single-stranded regions of nucleic acids and that dye sugar-phosphate chain does not seem to prevent adoption of the intercalation in short parallel double-helical regions with mixed puckered states of the riboses, but the requirement of noncomplementary base pairing is possible. These results may hydrogen bonding of proflavine on either chain of the anti- have some bearing on the strong binding of acridine dyes (3) parallel dimer seems to prevent it. This might be ultimately to denatured DNA, single-stranded RNA, and other single- related to the local symmetry and regularity of the dimer. In stranded polynucleotides like poly(A) (38), in which the for- the complementary antiparallel dimers, the dyad axis is per- mation of short parallel double-helical regions within one pendicular to the helical axis and the proflavine nucleic acid molecule or between two molecules is possible or could be complex can assume this symmetry because the drug possesses promoted by dye binding. One can also visualize dye interca- a dyad axis in its plane. The resulting hydrogen bonding of each lation in antiparallel chains involving non-Watson-Crick amino group of proflavine to the phosphates on either side base-paired sites. seemingly prefers the C3'-endo-C3'-endo puckering sequence Among the various biological effects induced by acridines, rather than the mixed puckering sequence. On the other hand, the ability to produce frameshift mutations rather than point in the noncomplementary parallel dimer of CpA the dyad axis mutations was crucial for the elucidation of the genetic code is parallel to the helical axis and hydrogen bonding to only one (39, 40). Several models have been proposed to explain phosphate oxygen is expected because proflavine does not frameshift mutagenesis (40-45). All these models rely on dye possess a dyad axis perpendicular to its ring plane. However, binding to DNA either by intercalation in double strands (41, disordering of the drug accomplishes hydrogen bonding of the 42) or by intercalation and stacking interactions in single strands drug to both chains of the CpA helix, but not simulta- (42-45). However, none of these models explains why a number neously. of intercalating agents that bind strongly to DNA possess a poor A comparison with the dApT double helical segments of the or nonexistent frameshift mutagenic potential (44-46). Thus, tetranucleotide dApTpApT (33), in which the same alternating the relationships between intercalation and frameshift muta- low-anti-C3'-endo, high-anti-C2'-endo sequence of glycosyl genesis (if any) are unclear. It is known that frameshift muta- torsions and ribose puckers is found, shows that this sequence tions tend to arise at strand discontinuities and in the vicinity of conformations is not sufficient for extending the interplanar of local base sequence redundancy (44, 45). If self-pairs of ad- distance from 3.4 A to 6.8 A. An examination of the other torsion enine or cytosine bases between parallel strands can be pro- angles of dApTpApT with the intercalated dimer structures moted by proflavine, it would be expected that such self-pairing (Table 1) shows that it is the rotation about C5'-05' bond (02 would also occur most easily between single-strand termini with angle) at the C2'-endo sugar that is responsible for increasing local base sequence redundancy. In this regard, it is interesting the interplanar spacing. Such a conclusion was also reached to note that methylation of the ring nitrogen abolishes muta- from computer modeling studies (35). genesis for An (except acriflavine), despite enhanced dye binding interesting point that has emerged from the present to DNA (46, 47). Such methylated drugs would be unable to crystal structure is that, in addition to the alternating pucker induce cytosine self-pairs by giving a proton to the pair or by sequence, there is also an alternation in the amplitude of sharing one. The upward shift of the pK of the nitrogen N3 in puckering, Tm. The C3'-endo ribose is much less puckered (Tm the triply hydrogen-bonded (C-C)+ pair allows the abstraction Downloaded by guest on September 25, 2021 1856 Biochemistry: Westhof and Sundaralingam Proc. Natl. Acad. Sci. USA 77 (1980) of the proton from the proflavine, which may provide a 22. Suck, D., Manor, P. C. & Saenger, W. (1976) Acta Crystallogr. chemical basis for the proposed alternative mechanism of B32, 1727-1737. frameshift mutagenesis. This might also explain the rough 23. Neidle, S., Taylor, G., Sanderson, M., Shieh, H. S. & Berman, H. correlation between the basicity of acridines and their muta- M. (1978) Nucleic Acids Res. 5, 4417-4422. genic effects (48, 49). 24. Albert, A. (1966) The Acridines (St. Martin's, New York), p. 158. We thank Dr. S. T. Rao for his generous help with the computer 25. Wrobel, A., Rabezenko, A. & Shugar, 0. (1970) Acta Biochim. programs used in this work. This research was supported by American Pol. 17,339-349. Cancer Society Grant CH-128 and the College of Agricultural and Life 26. Inman, R. B. (1969) J. Mol. Biol. 9,624-637. Sciences, University of Wisconsin-Madison. E.W. acknowledges a 27. Inman, R. B. & Baldwin, R. L. (1964) J. Mol. Biol. 8,452-469. Fulbright-Hays award from the Commission for Educational Ex- 28. Hartman, K. A. & Rich, A. (1965) J. Am. Chem. 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