Nucleic acid constituent interactions

Transcription

1 Proc. Indian Acad. Sci., Vol. 87 B (Experimental Biology-Z), No. 4, April 1978, pp. printed in India. Nucleic acid constituent interactions ROBERT REIN, RICK L ORNSTEIN and ROBERT D MACELROY* Department of Experimental Pathology, Roswell Park Memorial Institute, Buffalo, NY 14263, USA Department of Biophysical Sciences, State University of New York at Buffalo, Buffalo, NY 14226, USA *NASA-Ames, Moffett Field, California, USA MS received on 4 January 1978 Abstract. Hydrogen bonding contributes of the order of 5-15 kcal/rnol base pair to the stability of the helix (electronic or intrinsic energy). This contribution is selective, i.e., there is a preferential stability of the Watson-Crick G-C pair relative to all other pairs. Stacking interactions contribute approximately of the same order as hydrogen bonding. Perhaps the most interesting aspect of the stacking interactions which emerges from the theoretical analysis is the fact that the stacking maxima are not necessarily at the angles the successive base pair plans assume in a regular double helix. Consequently some sequence dependent structure peculiarities may arise. That is, the double helix may have a fine structure contingent on the sequence of base pairs. Indeed such sequence dependent polymorphism has been reported in the recent literature and appears to influence the ability of aromatic drugs to intercalate into the helix. The solvent effect which is another factor of stability seems to decrease somewhat bonding scheme preferences. For example, in the model we used to estimate solvent effect, we find that the G-C pair formation is de-stabilized strongly in water, while the A-T pair formation is mildly enhanced. The continuum model of solvent effect leads to similar qualitative conclusions. Studies of backbone conformation indicate that only a limited range of conformational states are comparable with the helical configuration. Improved empirical methods are needed in order to successfully calculate backbone effects for relatively large segments of nucleic acids. Keywords. DNA; interactions; speciflcity. 1. Introduction In the last decade there has been a great deal of interest in the secondary structure of nucleic acids. The secondary structure of nucleic acids, as well as other biopolymers, is conferred by weak intermolecular interactions between the constituent molecules as well as by intermolecular interactions with the environment. In the first section, this paper will describe some of the theoretical and experimental literature on the conceptual components i.e., hydrogen-bonding, base-stacking, backbone, and solvent energetics, that contribute to the overall structural and biological properties of nucleic acids. In the second section, recent calculations employing an empiricalpotential function with optimized parameters that have been used to predict basestacking (Ornstein et of 1978a) and intercalation (Ornstein and Rein unpublished work; Rein and Ornstein 1978b) are discussed. 2. Component interactions responsible for nucleic acid secondary structure and stability As with other non-covalent intermolecular interactions, a theoretical analysis of 135

2 136 Robert Rein et al nucleic acid interactions can be accomplished either by a supermolecular approach or by perturbation theory. In the former the interacting molecules are considered as a single system. A calculation of the energies of the separated constitutents follows and the difference of these quantities is taken to be the interaction energy. The obstacles which make it impractical to employ the supermolecular approach have been reviewed (Rein 1977). A major alternative to the supermolecular approach for treatment of the intermolecular interactions is based on the assumption that the individual interacting molecules preserve their individuality. That is, the molecular orbitals of the individual molecules can be utilized for construction of good zeroth order basis functions for a description of the perturbation of the interacting system. When the interacting molecules are sufficientlyapart their electronic systems are not significantly overlapping. The above assumption is indeed a very good approximation for the interacting systems Hydrogen bonding Calculated hydrogen bond energies by several different authors employing various levels of approximation of perturbation theory have recently been compared in detail (Rein 1977). These results can be summarized in terms of the relative orders observed (table 1). Experimental determination of the relative strength of various hydrogen bonding schemes are shown in table 2. It can be seen that the Watson-Crick hydrogen bonding forms are favoured over nonwatson-crick forms; although, the actual quantita- Relative ordering ofhydrogen bond energies by various methods of calcula Table 1. tion Reference Nash and Bradley (1966) Kudritskaya and Danilov (1976) Pullman and Pullman (1969) Rein et a1(975) Method Monopole-electrostatic approximation Atomic dipole approximation Monopole induced dipole approximation Multiple-bond polorizability approximation Order GC>GG ~ CC~GU,>UU> AG>AU>AC>CU>AA GC>GG>CC~GT~GU> AG>AC>AU>AT>AA> UU>TT>CU>CT GC>GG>CC ~ GT>AA> AC>AG>AU>CT>AT>TT GC ~ GU>AG>AT>AU> UU Table 2. Experimental results on the relative strength of hydrogen bonding Results Solvent Method Reference A-U>A-A or U-U CDCl 3 IR Hamlin et al (1965) CDCI 3 IR Kyoguku et al (1966) G-C>G-G or C-C CDCI 3 IR Katz and Penman (1966) G-C>A-T or A-U MEsSO IR Katz and Penman (1966) MEsSO NMR Shoup et al (1966) G-G>C-C CHCI 8 IR Kyoguku et al (1966) U-U>A-A CHCI 3 IR Kyoguku et al (1966)

3 Nucleic acid constituent interactions 137 Table 3. Decomposition of energies in hydrogen bonding Watson-Crick base pairs (kcal/mole) Reference Pair EEl Epol Edisp Erep Etotal Kudritskaya and Danilov (1976) A-T '6-0,7-7 0 Egan et al (1974) A-T - 9'0-1'1 -Jot '0 Claverie (1968) A-T - 6' Kudritskaya and Danilov (1976) A-V ,7-0,9-7-3 Egan et al (1974) A-V - 9'7-1'5-0' Pullman and Pullman (1969) A-V ,3-0,7-5-6 Kudritskaya and Danilov (1976) G-C -14,1-1'9-0,8-16,8 Egan et al (1974) G-C -22' ' ,5 Claverie (1968) G-C -21' '0 Pullman and Pullman (1969) G-C '0-1'3-19'2 tive differences are not that large. The G-C pair being the most stable. In spite of the fact, that we have compared in vacuo hydrogen bond energies with solvent experiments, the overall degree of comparison is quite good. It is interesting to note that the simple electrostatic model calculations of Nash and Bradley (1966) agree with the experimental results as well as any of the more refined methods. This can be rationalized by a comparison of the component interactions presented in table 3. Thus we observe that the repulsive component tends to cancel the second order components i.e., polarization and dispersion, leaving the electrostatic term as the dominant one Base-stacking Analysis offorces: Base-stacking is an important feature of the stability of helical nucleic acids. Stacking interactions are observed in polynucleotides as well as in aggregates of the various base fragments (Bugg et al 1971; Ts'o 1970). An understanding of the forces involved in base stacking interactions is essential to our understanding of the stability and structural features of nucleic acids. The pioneering calculations of DeVoe and Tinoco (1962) employed a relatively simple approximation employing London's theory. More refined approximations have been extensively used (Rein 1977; Rein et ai1969). We have recently applied, perhaps, one of the most refined perturbation approximations employing a segmental multipole-multipole approximation of the electrostatic component with similarly refined second-order terms so as to investigate the relative importance ofthe various component interactions. These calculations (Ornstein, unpublished) indicate that the second order components are responsible, in general, for most of the stability arising from stacking interactions, however, they show little dependence on angular rotation of one pair with respect to the other pair about the helix axis. The repulsive component is comparatively insignificant in magnitude because of the relatively large interplanar pair separation. Although the electrostatic component is intermediate in magnitude, it plays the greatest role in determining the location of stacking minima as a function of angle of rotation. This is clearly shown in figures I and 2 for the stacking sequences t G.C/G.C t and t A.TJA.T t. These results agree qualitatively with numerous

4 138 Robert Rein et al.. '0 E<, '0 u x: '" Cl L 12.00r ~ _._ E _.-_._.... _ ~ ' ~.:<.:.=_..! :.>( ~ Angle of rotation Figure 1. Interaction energy: (0- CfO-- Cl. 1. dispersion; 2. polarization; 3. repulsion; 4. electrostatic; 5. total r ,.!! 6'00 o ~ '0 u:>t. o >, Cl L ~ -6,00 llj ,,., ~.,---<, -,.: :::::-.".,.;;,."'~.;;;.;;../.<.... ~.:~.x ~, '-_--J. -L-.l.-_-J'--_-L...L- L-_---J o Angle of rototion Figure 2. Interaction energy (A - TIA- T). 1. dispersion; 2. polarization; 3. repulsion; 4. electrostatic; 5. total. less refined perturbation treatments (Kudritskaya and Danilov 1976; Fugita et al 1974; Polozov et a/1975) Base-stacking dependent secondary structure A wide body ofexperimental data suggest a more than casual relationship between DNA base sequence and secondary structure. For example, Bram and coworkers (Borer et al 1974; Bram 1971) report a variation in the x-ray pattern for natural B-form DNA's, while Arnott and coworkers (Arnott and Hukins 1973; Arnott et al 1974a); Arnott and Selsing 1974a,b, 1975; Arnott 1977) and Bram and coworkers (Bram and Tougard 1972; Bram 1973)report that their x-ray studies on nonrandom, synthetic DNA's (with known base-sequence) indicate a strong dependence of secondary structure and base sequence. Based on x-ray as well as a variety of solution

5 Nucleic acid constituent interactions 139 data, it appears that the base-stacking pattern in DNA is a function of base sequence. It has been suggested that this type of structural variation, at least in part, may provide the necessary recognizable features required for genetic regulation and enzyme specificity. Moreover, recent experimental (Krugh and Reinhardt 1975; Tsai et al 1977; Sobell et al 1977) and theoretical (Pack and Loew 1977; Pack and Loew 1977 unpublished) evidence suggests that base interactions are important in determining the observed preferences of intercalation specificities. Most theoretical investigations of base-stacking have assumed that all sequences of base-pairs align themselves as in the standard B-DNA configuration. Based on available experimental evidence it is, however, apparent that this is not the case; although the B-DNA configuration is apparently a reasonable' average' structure for the various sequences of stacked base-pairs. Using our refined perturbation method which employs, for instance, the segmented multipole-multipole approximation electrostatic component, we recently located the stacking minimum for the various sequences as a function of base-pair separation and angle of rotation(ornstein 1978). Table 4 shows these values for the three different composition groups. These calculations indicate the most stable position within the range of 36±30 for the angle of rotation. Table 4. Preferred stacking locations' for different composition sequence isomers Preferred stacking location Composition Mini-helix Energy (kcal/mole) Angle of rotation Base-pair Group (degrees) (Separation in A) t C G r -16'53" 32 3'1 I G C G,C tgg! -12, t! -10, I a C t TA! I A T A,T tat! - 6' la T a,c,a.t tat I I TAt 1~ ~! '2 t C G I -10, I TAt 1~ ~! t G C I I A Tt 'Range of scan from 36±30 in 2" intervals and separation in nm intervals. zarrows indicate IUPAC convention from C3' to C5' (interval) on either side of phosphodiester linkage. "Underlined values are global minima.

6 140 Robert Rein et al It is indeed interesting that for five out of the ten unique sequences, the global minima in stacking energies occur within the range and 0, nm. These five global minima are for all possible locations i.e., angular rotations from Only one such global minimum is observed for the G, C composition group, while two minima are observed for the A, T and mixed compositiongroups. This suggests that the former group offers a greater opportunity for sequence dependent structural variation than the others. We shall return to a discussion of base-stacking interactions in section Solvent effects on nucleic acid stability Most organic solvents denaturate DNA. Water seemed to be unique in stabilizing the double helical form. Sinanoglu and Abdulnur (1964), using continuum reaction field theory, were the first to analyse the effect of solvent on helix-coil transition. The results of Sinanoglu and Abdulnur (1964) were successful in qualitatively explaining the observed solvent effect for water and a number of organic solvents. However, the solvent effect values in water of 14 kcaljrnol for GC dimer and -19 kcal/mol for AT dimer formation are too large to be reconcilable with thermodynamic values for these processes. A second approach, emphasizes the importance of specific sites on the solute molecule where properly oriented solvent molecules can be preferentially attached. In this approach the basic entity investigated is the supermolecule, i.e., the complex of solute molecule with the solvent molecules attached to it. Alagona et al (1973) developed this approach for the hydration of formamide, and Port and Pullman (1973) performed a similar study for adenine, guanine, cytosine and thymine. Port and Pullman concluded that for the four bases the most favourable positions for hydration are around the base peripheries with the oxygen of water in the molecular plane. Out of the plane, monohydration appeared unlikely in the case of the bases. The above studies on the DNA bases have not yet considered the question ofwhether the stacked or hydrogen bonded configuration is preferred in a given solvent, e.g. water. Studies in this direction are in progress in our group. To calculate the solvent effect on base pair formation, Rein et al (1975) assumed that the solvent effect arises principally due to the dehydration of the bases accompanying pair formation. The hydration sites on the bases are the same that will enter base-base hydrogen bonding. Thus the number of water molecules displaced are three for G and C and two for A and T, respectively. The dehydration energies are assigned values by judicial choice from the hydration energies calculated by Port and Pullman (1973). The solvent effect is calculated according to the formula below which can be considered a simplified version of the method of Poland and Scheraga (1965). Esolvent eff = Ebase pair.. nh 20 - (Ebase 1...n/H 20 + Ebase 2. n"h 20). These calculations gave a solvent effect on the GC pair formation of 7 kcal/mol and --2kcal/mol for the AT pair formation. Using these values together with calculated hydrogen bond stacking and conformational adjustment energies, the authors have obtained a qualitative agreement with the experimentally determined thermodynamics data of Borer et al (1974). Based on these results it would seem that the dehydration model is useful for explaining the solvent effect in helix formation.

7 Nucleic acid constituent interactions Backbone energetics The polynucleotide backbone interaction energy is as important in the stabilization of a polynucleotide helix as base-pairing (hydrogen bonding) and base-stacking energies (Sundaralingam 1975). In general, most of the backbone structural diversity arises from rotations around the sugar-backbone C(3')-C(4') bond and the internucleotide P-O bonds, thus suggesting a 'rigid' nucleotide unit (Sundaralingam 1975). Despite considerable differences, 19 known common 5'-nucleotide structures exhibit an overwhelming preference for C(2')-endo and C(3')-endo type conformations; thus indicating the essential role played by short range intramolecular forces. As a rule, only anti-glycosidic conformations are observed for polynucleotides. The structural properties of nucleic acids have been investigated by two methods of calculation: (i) molecular-orbital, and (ii) perturbation procedures. Analysis of relatively large nucleic acid fragments remain an expensive undertaking using the former procedures. Perturbation procedures can be divided into theoretically, rigorous' or empirical methods. For nucleic acid segments of a size equal to or larger than a miniature or mini-helix, only the empirical methods are practical. In an extensive review, Pullman and Saran (1976) have recently compared the various methods and have indicated the generally unsatisfactory results of the empirical methods available at or before It is now obvious that at least part of the problem with empirical techniques is their lack of correspondence with more refined perturbation methods. In this direction, we have recently constructed an empirical potential functions with optimized parameters. In the next section we briefly discuss some of our recent calculations with this potential. 3. Improved empirical-potential function calculations of some nucleic acid properties Most of the theoretical studies on nucleic acid secondary structure have centered around various intermolecular interaction schemes which are essentially applications of Raleigh-Schrodinger perturbation theory. A detailed description of the interaction components i.e., electrostatic, polarization, dispersion, and short-range overlap repulsion, employed in intermolecular interaction calculations has appeared in a series of papers from our group and has recently been reviewed (Rein 1977, Rein 1973, Rein 1975). Due to the relatively large size of nucleic acid segments of interest, for example, a miniature or mini-helix, one is from a practical standpoint often forced to employ approximations ofperturbationtheory thathave become known as classical or empirical methods. The fundamental differences among these schemes lie in the levels of approximation employed- i.e., levels of expansion, wave functions and atomic densities, parametrization, etc. Empirical methods, however, have often led to erroneous results (Pullman and Saran 1976) and non-uniformity when applied to nucleic acid conformational problems Empirical potentials with optimizedparameters: Base-stacking We have recently completed a comparative study of the various schemes as applied

8 142 Robert Rein et al to base-stacking interactions (Ornstein et ai1978). Since the electrostatic component is known (Kudritskaya and Danilov 1976; Claverie 1968; Polozov et al 1975) as the most influential component of the stacking interaction, we investigated its relationship in depth. Atomic charges computed with IEHT, CNDO/2, Del Re separation, and ab initio wave functions were employed in a monopole-monopole approximation of the electrostatic component and compared to a segmented muitipole-muitipole approximation. The angular minimum predicted by the segmented muitipole-muitipole approximation is expected to be the most reliable estimate. However, the segmented muitipole-muitipole approximation procedure is many times more expensive to calculate than the simpler monopole-monopole approximation values. Thus, from a practical standpoint, it is important to determine which set of monopole charges best agree with the segmented multipole-multipole approximation predictions. Figure 3 shows the comparison for the t C'G/G'C t sequence. Electrostatic values were computed at 10 intervals of rotation about the helix axis with the base-pair separation held fixed at 0'34 nm. As indicated in figure 3, the IEHT and CNDO/2 charges in the monopole-monopole approximation are about equal in their ability to predict the location and magnitude of the electrostatic minima as predicted by the segmented muitipole-muitipole approximation. The magnitude of minima energies predicted by the Del Re separation charges are, in general, three to four times more in error with segmented muitipole-muitipole approximation predictions, than are the energies determined by IEHT or CNDO/2 charges. The ab initio charges are least able to predict the segmented multipole-multipole approximation trends. In attempting to explain the poor agreement with ab initio charges, we note that several atomic centers have negative charges in excess of O Se r , ,"'--... /'_..._"... I 4~,,I -6'00 -g OOL-_.L-_..L-_.J-_...L._...l-_-L-_-l-_---'--_-.l.._--' o Angle of rototion Figure 3. Electrostatic component of stacking interactions as a function of angle of rotation for the dimer t g..~ t monopole-monopole approximations (MMA) are compared for sets of charges determined by the following molecular-orbital methods: (1) IEHT (Sob~lI et ai1977); (2) Del Re separation (Renugopalakrishnan et ai197l); (3) CNDO/2 (Giessner-Prettre and Pullman 1968); (4) ab initio (Clementi 1969); (5) Del Re separation (Berthod and Pullman 1965)and (6) ab initio (Mely and Pullman 1969). A segmental multipole-multipole approximation employing the IEHT method is noted by the curve 7.

9 Nucleic acid constituent interactions 143 We similarly compared more refined expressions for the polarization, dispersion and repulsive components with those often used in empirical potential functions. We thus constructed a complete function with optimized parameters and found that base stacking results determined by this method compare as well with melting and x-ray data as do any of the more refined perturbation methods Empirical potentials with optimized parameters: Energetics of intercalation Using a similar empirical potential functions with optimized parameters, we recently investigated the role of base-base (stacking) interactions, base-phosphate interactions, base-sugar interactions, and ionic charge (on phosphate group) in determining the sequence specificity of intercalation for all complementary dinucleoside triphosphate mini-helices. The calculated energies predict all experimentally observed trends in sequence specificity regarding structural transitions from the B-DNA to the intercalated-complex conformation. A comparison of the total energies required to make this structural transition for the various mini-helices are shown in table 5. Within the three classes of composition isomers (G/C, A/T, mixed), the pyrimidine (3'-5') purine sequence mini-helices are energetically preferred over the other helices to make the change from the B-DNA to the intercalated conformation. The purine (3'-5') pyrimidine sequence helices are always least preferred. These trends agree with Table 5. Total energy- to change from B-DNA2 to intercalated-complex'' conformation for complementary dinucleoside triphosphates with ionized or neutralized phosphate groups. G, C Composition isomers Ionized' Neutralized' G.C/C.G.5 17'()6 12'2 C.G/C.G '4 G.C/G.C 24' C.G{G.C 30'6 20'6 A, T composition isomers A.T{T.A '5 T.A{T.A '5 A.T/A.T '3 T.A/A.T 24' Mixed composition isomers G.C/T.A '4 A.T/C.G 17' T.A/C.G G.C/A.T 22'8 16'4 C.G{T.A A.T{G.C T.A{G.C C.G{A.T "Ihe total energy to change from the B-DNA to the intercalated conformation is comprised of the sum of differences between the component interaction for the intercalated minus the B-DNA form. The components are BB, BP, BS, PP, PS, SS, and torisonal potentials, 2B-DNA conformation from x-ray study of Arnott & Hukins (1972). 3Intercalated-complex conformation from x-ray study of Tsai et al (1977). 'Energies determined in this study with empirical partitioned function. 5The following short-hand notation is employed in this table: _ tg.ci G.C{C.G - I C.G.j., etc., where the arrows indicate (internal) convention of direction from the C3' atom of one deoxyribose to the C5' atom of the next deoxyribose. -Energies in kcal/mole.

10 144 Robert Rein et at available x-ray and solution evidence (Krugh and Reinhardt 1975; Tsai et al 1977; Sobell et a/1977) as well as with the CNDO and PCILO calculations (Pack and Loew 1977a.b). We note that base-base energies are always responsible for at least half of the difference in energies between isomeric pyrimidine (3'-5') purine and purine (3'-5') pyrimidine sequences. Base-phosphate interactions constitute most of the remaining energy difference when the phosphate groups are ionized, but play a much reduced role when the phosphate groups are neutralized. Acknowledgements This research was supported partially by NASA-Ames University Consortium interchange NCA~-OR and Grant NSG-3705 from the National Aeronautical and Space Administration and also by Core Grant No. CA from the U.S Public Health Service. The generous allotment of time by SUNY/AB Computer Center is greatly appreciated. References Alagona G, Pullman A, Serocco E and Tomasi F 1973 Int, J. Peptide Prot, Res Arnott S 1977 in Proceedings ofthe First Cleveland Symposium on Macromolecules ed. A G Walton (New York: Elsevier) p. 87 Arnott S et al1974a J. Mol. Biol Arnott Sand Hukins D W L 1973J. Mol. BioI Arnott S and Selsing E 1974a J. Mol. BioI Arnott S and SeIsing E 1974b J. Mol. Biol Arnott S and SeIsing E 1975J. Mol. Biol Berthod H and Pullman A 1965 J. Chim. Phys Borer P N, Dengler B, Tinoco I Jr and Uhlenbeck D C 1974 J. Mol. Biol Bram S 1971 Nature New Biol Bram S 1973Proc. Not. Acad. sa. USA Bram Sand Tougard P 1972 Nature New Biol Bugg C E, Thomas J M, SundaraIingam M and Rao S T 1971 Biopolymers CIaverie P 1968 Molecular Association in Biology ed. B Pullman (New York: Academic Press) p. 115 Clementi E et Acta Phys, Acad. Sci. Hung DeVoe Hand Tinoco I Jr 1962 J. Mol. Bioi Bgan J T, Swissler T J and Rein R 1974 Int, J. Quantum Chem: Quant, Biol. Symp, no Fugita H, Imamura A and Nagata C 1974 J. Theor. Bioi Gibson K D and Scheraga H A 1967 Proc. Nat. Acad. sa. USA Giessner-Prettre C and Pullman A 1968 Theor. Chim. Acta Hamlin R M, Lord R C and Rich A 1965 Science Katz K and Penman S 1966 J. Mol. Bioi Krugh T Rand Reinhardt C G 1975 J. Mol. Bioi Kudritskaya V K and Danilov V I 1976 J. Theor, Bioi Kyoguku Y, Lord R D and Rich A 1966 Science Mely B and Pullman A 1969 Theor. Chim. Acta Nash H A and Bradley D F 1966 J. Chem. Phys Ornstein R, Rein R, Breen D and MacEIroy R 1978 Biopolymers (in press) Ornstein R 1978 Ph.D Thesis (to appear) Ornstein R and Rein R 1978 Biopolymers (submitted) Pack GRand Loew G 1977 Int, J. Quantum Chem.: Quant. Biol, Symp, (in press)

11 Nucleic acid constituent interactions 145 Pack GRand Loew G 1977 Biochim. Biophys. Acta (submitted) Poland DC and Scheraga H S 1965 Biopolymers Polozov R V, Poltev V I and Sukhovukov B I 1975 J. Theor. BioI Port G N J and Pullman A 1973 Theor. Chim. Acta (Berl.) Pullman B and Pullman A 1969 Prog. Nucl. Acid. Res. Mol. Biol Pullman Band Saran A 1976 Prog. Nue!. Acid. Res. Mol. BioI Rein R 1973 Adv. Quant. Chem Rein R 1975 NATO Symposium in Quantum Chemistry eds J M Andre and J Ladik (New York: Plenum Press) p. 505 Rein R 1977 Perspectives in Quantum Chemistry ed, B Pullman (in press) Rein R, Coeckelelenbergh Y and Egan J T 1975 lnt. J. Quant. Chem.: Quant. BioI. Symp, Rein R et al 1969 Ann. N. Y. Acad. Sci Rein Rand Ornstein R 1978 Abstracts of Theoretical Chemistry Symposium, Indian Institute of Technology, Bombay 1977 (to appear) Renugopalakrishnan V, Lakshminarayanan A V and Sasisekharan V 1971 Biopolymers Shoup R R, Miles H T and Becker E D 1966 Biochem. Biophys. Res. Commun Sinanoglu 0 and Abdulnur S 1964 J. Photochem. Photobiol Sobell H, Tsai C C, Jain S C and Gilbert S G 1977 J. Mol. BioI Sundaralingam M 1975 in Structure and Conformation of Nucleic Acids and Protein-Nucleic Acid Interactions eds M Sundaralingam and S T Rao (Baltimore: Univ. Park Press) p. 487 Tsai CC, Jain S C and Sobell H M 1977 J. Mol. BioI Ts'o POP 1970 in Fine Structure ofproteins and Nucleic Acids eds G Fasman and Timisheff (New York: Marcel Dekker) p,