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Interactions of 6-thioguanine in B-DNA: Possible mechanism of its mutagenic action
J. theor. BioL (1975) 54, 167-174
Interactions of 6-thioguanine in B-DNA: Possible Mechanism of its Mutagenic Action H. CHOJNACKI AND W. A. SOKALSKI
Department of Physical Chemistry, Institute of Organic and Physical Chemistry, 50-370 Wroclaw, Wybrzef:e Wyspianskiego 27, Poland (Received 4 April 1974, and in revisedform 10 October 1974) Perturbation energies calculated for complementary base systems containing 6-thioguanine have been compared with the relevant results for normal base pairs. It is concluded that a single 6-thioguanine incorporated into B-DNA results in the increase of horizontal and vertical interactions whereas the respective stacking interactions between the same neighbours tend to decrease. The mutagenic action of the 6-thioguanine molecule in the DNA is interpreted as inhibiting of transcription and replication processes.
1. Introduction
The mechanism of the antitumour activity of 6-thioguanine has not yet been completely established. However, it is thought that the inhibitory action of this compound is a result of its incorporation into DNA replacing the normal guanine component (LePage, 1960; Kaplan, Smith & Tomlin, 1961; LePage & Jones, 1961; LePage & Junga, 1963). Related to this, Mautner & Jaff6 (1961) suggested that 6-thioguanine is able to form rather unusually strong hydrogen bonds with cytosine leading to interference of the replication process of DNA. Furthermore, some evidence has been presented indicating that sulphur containing molecular systems may interact more strongly than their oxygen analogues (Flett, 1953; Zackrisson, 1961; Niedzielski, Drago & Middaugh, 1964; Krueger, 1970; Snyder, Schreiber & Spencer, 1973). On the other hand, intermoleculer N - - H - . . S hydrogen bonds are considered to be weaker than the usually shorter bonds of the N - - H - - . O type (Donohue, 1969; Lutsky & Goncharova, 1972; Geller, Pohorille & Jaworski, 1973). Thus, the experimentally determined cases of stronger interactions of sulphur complementary base analogues are explained by increased vertical interactions (Scheit & Faerber, 1971; Faerber, Scheit & Sommer, 1972). Recent studies based on the comparative X-ray studies of 6-thioguanine, 6-thioguanosine and guanine (Bugg & Thewalt, 1970; t67
168
H. CHOJNACKI
AND W.
A. S O K A L S K I
Thewalt & Bugg, 1972) concluded that antineoplastic activity of 6-thioguanine was mainly due to alterations of hydrogen bond lengths of the respective base pairs, a study which seems to indicate that vertical interactions play a minor role. The controversial views mentioned above were an encouragement to compare theoretical interaction energies for 6-thioguanine and guanine base pairs of the B-DNA system.
2. Outline of the Calculation Method
The relative interaction energies have been calculated within the framework of the Rayleigh-Schr6dinger perturbation theory with the use of semiempirical parametrization of the LCMO method (Chojnacki & Sokalski, 1973). According to this approach the intermolecular interaction energy is given by E = E I + E II (1) where E ~ and E n are the first and second order perturbation energies, respectively. When intermolecular exchange effects are neglected the first order perturbation energy may be approximated to
= E Z (P,e,r,,- Pr Y
-- e, V,b + ZoZ /Ro )
S
representing an electrostatic term (Fujita, Imamura & Nagata, 1974; Fukui & Fujimoto, 1968). Neglecting dispersion and polarization forces the second order interaction energy becomes
ff ..o .f g} (z ~i
~"
(31 i
j.
E°-E~
resembling the charge-transfer (delocalization) contribution in the total interaction energy (Fukui & Fujimoto, 1968). The indices r and s denote atomic orbitals, a and b correspond to atomic centers, whilst the i and j are molecular orbitals belonging to the interacting molecules R and S, respectively at the R,b interatomic distance. Eigenvalues Ek, eigenvectors cut and electron populations P~ were evaluated by the use of the standard CNDO/2 approximation (Pople & Segal, 1966). The two centre Coulomb integrals F,~ and nuclear attraction integrals V,o and V,b were calculated with the ns Slater atomic orbitals. The resonance integrals H,s were approximated by the Wolfsberg & Helmholz (1952) formula H,s = 0.5K(H,, + Hs.~)S,. ~ (4)
INTERACTIONS OF 6-THIOGUANINE IN B-DNA
169
with K = 1 and the respective valence state ionization energies Hkk assumed according to Hinze & Jaff6 (1962) or in the case of a sulphur atom, taken from Levison & Perkins (1969). Overlap integrals S,. s were evaluated with the Slater basis set. The d atomic orbitals of sulphur were not considered explicitly as they do not seem to confer any significant contribution to the interaction energy (Guimon, Gonbeau & Pfister-Guillouzo, 1973). The method used permits a partitioning of the E n energy into tr--tr and re--re contributions. However, the partitioning is precise only in the case of coplanar systems since the overlap integrals were not separated into tr--o" and ~--rc components.
(-722) + 260
~-,53) -n
+~ ~
\~,,9' I
+~
~-"
/
r
f : ~ (- 646)
" -30
%.
\
/
+43
6
" ~ - -...~
II
#
(- 3
(~
rE) . . . . 3-3o~
~
(-624) |
+133
1
+6
+'~
\
/ ~
+,6
(--347) / /
\_
(+!87) +175
~
+ 6
....
( + 2 2 _ 2 ) ~ • 191 ~ J ~
1
- / ';
-'
./
+~"., (- 640) +471
+,4o
A -I- 135 H{~,)~l- 149
F1G. 1. Molecular geometry assumed with the respective charge distribution (milliproton units) of the 6-thioguanine-cytosine base pair calculated for isolated molecules by CNDO/2 method. The non-bracketted values are a-electron charges and those in parentheses re-electroncharges, respectively.
The geometry of the 6-thioguanine-cytosine system (Fig. 1) assumed in the calculation resembled that of the guanine-cytosine base pair of the WatsonCrick configuration. The N - H . • • S and C = S bond lengths were taken equal to 3.30 • and 1.78/~ respectively, instead of 3.00/~ and 1.20/~ for the normal guanine-cytosine base pair (Blizzard & Santry, 1969). The interplanar distance between neighbouring purines was assumed to be 3.38 A with the relative rotation of the components by 36 ° around the helix axis (Fig. 2) in accordance with the X-ray diffraction data for B-DNA (Arnott, Dover & Wonacott, 1969).
170
H. CHOJNACKI A N D W . A . SOKALSKI
FIG.2. Base stacking patterns in the B-DNA crystal fibres (Arnott et at., 1969) as viewed perpendic~ilarto the plane of the lower base represented by dashed lines. The cross is centred on the position of the helix axis.
3. Results and Discussion
The interaction energies calculated for 6-thioguanine-cytosine, guanineguanine, 6-thioguanine-guanine and 6-thioguanine-6-thioguanine systems partitioned into components are given in Tabb 1. In accordance with experimental information (Shoup, Miles & Becker, 1966; Kyogoku, Lord & Rich, 1969; Scheffler & Sturtevant, 1969; Pohl, 1973) and earlier theoretical calculations (Pullman & Pullman, 1968; Bertran, 1972; Danilov, Zheltovsky & Kudritskaja, 1973; Nagata, Imamura & Fujita, 1973; Fujita, TABLE 1 Partitioning of intermolecular interaction energies (kca1Jmol) for complementary brrses and their 6-thioguanine analogue Molecular system Adeninethyminet Guanine-cytosinet 6-thioguaqine-cytosinc
t Chojnacki & Sokalski (1973).
-5.95 -12.78 - 17.25
-13.25 -13.74 -18.76
-04010 -0.0007 -0~0011
-
-19.20 -26-52 -36.01
I N T E R A C T I O N S OF 6 - T H I O G U A N I N E IN B - D N A
171
Imamura & Nagata, 1974; Rein, 1974) the calculated interaction energies for guanine--cytosine are greater than those of the adenine-thymine system. The observed enthalpy changes for base pairs formation at physiological conditions are -7.90 + 0.14 kcal/mole for dA.dT base pair (Schettler & Sturtevant, 1969) and -11.1 + 0.5 kcal/mole for dG.dC base pair ~ohl, 1973). In our theoretical approach, neglecting exchange, dispersion and polarization terms seems to be a rather drastic assumption. The role of neglected terms for small hydrogen bonded systems has been pointed out recently (Kollman & Allen, 1972; Daudey, 1974). However, at the equilibrium geometry of different molecular pairs the repulsive exchange contribution and the attractive dispersion and polarization terms tend to cancel each other (or tend to be constant) leaving the relative ordering of stabilization energies to be determined by the Coulomb and charge transfer interactions (Person, 1973). Hence, the limited success of simple electrostatic or charge transfer theories used in the past for description of small hydrogen bonded systems and typical charge transfer complexes respectively, may be justified. For large hydrogen bonded systems the charge transfer interactions seem to be more important (Malarski, 1974). On the other hand the charge transfer stabilization energy allows one to obtain a good correlation with observed heat of formation for tetracyanoethylene molecular complexes with various aromatic hydrocarbons (Herndon & Feuer, 1968). Therefore, we decided to use the sum of both contributions as a relative intermolecular energy for similar large molecular systems at equilibrium distances. However, the quantities calculated here in any case cannot be considered as absolute values of stabilization energy. The most advanced studies done hitherto within the scheme of perturbation theory do not reproduce properly potential energy curves even for small hydrogen bonded systems (Van Duijneveldt, 1969; Daudey, 1974). It is because of great mathematical complications in solving the intermolecular interaction problem reviewed recently by Certain & Bruch (1972). It is dit~cult to estimate the role of the solvent in the interaction energy as well as possible changes in the electronic structure of the complementary bases caused by neglecting the sugar moieties. The sulphur orbitals were assumed to have the same hybridization as the oxygen atom. However, the density maps obtained for the thiophene molecule indicate that atomic orbitals on sulphur are, in comparison with oxygen in the furan molecule, only slightly hybridized (M~trtensson & Chojnacki, 1973). Thus, the lone pair of sulphur is more diffuse than the oxygen atom lone pair resulting in a decrease of the overlap integral between sulphur and the hydrogen atom in the hydrogen bond. As seen from Table 1, the changes in the horizontal
172
H.
CHOJNACKI
AND
W.
A. S O K A L S K I
interaction energy upon replacing oxygen by sulphur atom in the systems looked at, are mainly due to charge transfer interactions as was found from IR measurements of similar systems (Malarski, 1974). It is concluded that the relative strengths of hydrogen bonds of the N - H - " • S and N - H . • • O type depend on the molecular geometry and electronic structure of interacting systems and must be considered individually. Assuming that, in spite of approximations made in the calculation, the qualitative relation between theoretical interaction energies still gives the higher horizontal interaction energy for the 6-thioguanlne-cytosine pair than that of the normal guanine-cytosine pair. Thus, 6-thioguanine incorporated into DNA may exert an inhibiting action on replication and transcription processes associated with the respective breaking of intermolecular hydrogen bonds. The results obtained indicate the importance of vertical interactions as well. In this case dispersion and polarization forces may contribute a greater share to the stabilization energy (Bertran, 1972; Danilov et al., 1973). The stacking interaction energies ordered as 6-thioguanineguanine > guanine-guanine > 6-ttfioguanine-6-thioguanlne indicate that vertical interactions for single molecules of 6-thioguanine incorporated into DNA should increase in comparison to the pertinent interactions of guanine. The destabilizing stacking interactions found for two adjacent 6-thioguanine molecules may lead to a distortion of the secondary structure of D N A at the incorporation site. In the single step of the replication process, a deficiency of energy at the region of replicase enzyme may appear. It would be the case when the energy gained from hydrolysis of nucleosidetriphosphate together with that of formation of hydrogen bonds and stacking interactions in the region of the new formed polynucleotide chain is less than the energy needed to cleavage the stronger hydrogen bonds in 6-thioguanine-cytosine pair together with the binding energy nucleosidemonophosphate to polynucleotide chain. Such a situation is more probable in transcription than replication where in the single step of the process, breaking of the two hydrogen bonds is involved. Other support of the supposition comes from the observation that the inhibitory action of 6-thioguanine is more pronounced in the RNA than in the DNA synthesis (Mandel, Latimer & Riis, 1965). It does not mean, however, that the incorporation of 6-thioguanine into nucleic acid has in every case led to lethal mutation since incorporation process into R N A has been completely inhibited only in the presence of blocking transcription actinomycin D (Kwan, Kwan & Mandel, 1973). It is concluded that mutagenic effects of 6-thioguanine may be mainly due to its incorporation into DNA although its incorporation into various fractions of R N A has been determined also (Gray & Rachmeler, 1967; Kwan et al., 1973). The confirmation of the above
INTERACTIONS OF 6 - T H I O G U A N I N E IN B-DNA
173
hypothesis derives f r o m studies o n t u m o u r s susceptible o r resistant t o the 6-thioguanine action. I t was e s t a b l i s h e d t h a t in susceptible t u m o u r s , 6-thioguanine is i n c o r p o r a t e d m a i n l y into D N A a n d into R N A in resistant lines (LePage & Junga, 1963). The numerical computation performed in part by Professor O. M ~ t e n s s o n at the Quantum Chemistry G r o u p o f the Uppsala University is warmly acknowledged. This work has been sponsored in part by the Polish Academy of Sciences under the P A N - 3 contract. REFERENCES ARNOYr, S., DOVER, S. D. & WONACOrr, A. J. (1969). Aeta crystallogr. B25, 2192. BERTRAN,J. (1972). J. theor. Biol. 34, 353. BLIZZARD,A. C. & SAr~TRY,D. P. (1969). J. theor. BioL 25, 461. BUGG, C. E. & THEWALr, U. (1970). J. Am. chem. Soc. 92, 7441. CERTaiN, P. R. & BRUCH, L. W. (1972). M T P lnt. Bev. Sci. Phys. Chem. 1, 113. C-'nOJNACKr,H. & SOKALSrd,W. A. (1973). J. molec. Struct. 15, 263. DANILOV,V. I., ZHEL'rOVSKY,N. V. & KODmrSr,~JA, Z. G. (1973). Preprint ITP-73-109R, Kiev. DAUDEY, J. P. (1974). Int. J. Quantum Chem. 8, 29. DONOrlUE, J. (1969). J. molec. Biol. 45, 231. FAERBER,P., SCHEIT,K. H. & SOMMER,H. (1972). Eur. J. Biochem. 27, 109. FLEa'r, M. S. C. (1953). J. chem. Soc. 347. FUJ1TA,H., IMAMURA,A. & NACArA, C. (1974). J. theor. BioL 45, 411. FUKUI, K. & FUJIMOTO,H. (1968). Bull. chem. Soc. Japan 41, 1989. GELLER, M., POHORILLE,A. & JAWORSKI,A. (1973). Biochim. biophys. Acta 331, 1. GRAY, P. N. & RACHMELER,M. (1967). Biochhn. biophys. Aeta 138, 432. GUIMON, C., GOr,rBEAU,D. & PFXSTER-GUILLOUZO,G. (1973). J. molec. Struet. 16, 271. HERNDON, W. C. & FEUtR, J. (1968). J. Am. chem. Soe. 90, 5914. I-IIr~ZE,J. & JAFrd, H. H. (1962). J. Am. chem. Soc. 84, 540. KAPLAN,H. S., SMrrH, D. C. & TOMUN, P. (1961). Nature, Lond. 190, 794. KOLLMAN,P. A. & ALLEN, L. C. (1"972). Chem. Rev. 72, 283. KRUEGER, P. J. (1970). Tetrahedron 26, 4753. KWAN, S. W., KWAN, S. P. & MANDEL,H. G. (1973). Cancer Res. 33, 950. KYO~OKU, Y., LORD, R. C. & RICH, A. (1969). Bioehim. biophys. Acta 179, 10. LEPAC;E,G. A. (1960). Cancer Res. 20, 403. LEPAGE, G. A. & JON~, M. (1961). Cancer Res. 21, 1590. LEPAGE, G. A. & JUNGA,I. G. (1963). Cancer Res. 23, 739. LEVISlON,K. A. & PERKINS,P. G. (1969). Theor. chim. Acta 14, 206. LUTSKY,A. E. & GONCHAROVA,E. I. (1972). Ukr. khim. Zh. 38, 1223. MALARS~, Z. (1974). Roczn. Chem. 48, 663. MANDEt., H. G., LA~MER, R. G. & PallS, M. (1965). Biochem. Pharmac. 14, 661. M~,RT~NSSON,O. & CHOJrqACKI,H. (1973). Actaphys. poL A44, 259. MAUTNER, H. G. & JAFFa, J. J. (1961). Biochem. Pharmac. 5, 343. NAGATA,C., IMAMURA,A. & FUJITA, H. (1973). Adv. Biophys. 4, 1. NmDZmLSKI, R. J., DRACO, R. S. & MIDDAUOH, R. L. (1964). J. Am.chem. Soc. 86, 1964. PERSON, W. B. (1973). In Spectroscopy and Structure of Molecular Complexes (J. Yarwood, ed.) p. 15. London: Plenum Press. POHL, F. M. (1973). Eur. Jr. Biochem. 42, 495. POPLE, J. A. & SEOAL,G. A. (1966). J. chem. Phys. 44, 3289. POLLMAN,A. & PULLMAN,B. (1968). Adv. Quant. Chem. 4, 267.
174
H. CHOJNACKI AND W. A. SOKALSKI
R~IN, R. (1974). Proceedings of Advanced Study Institute on Electronic Structure of Polymers and Molecular Crystals, Namur, Belgium. SCnr.~n~LER,I. E. & STtmTEVANT,J. M. (1969). J. molec. BioL 42, 577. Scmwr, K. H. & F.~'U~ER, P. (1971). Fur. J. Bioehem. 24, 385. SHotrP, R. R., M n ~ , H. T. & BECKER, L. D. (1966). Biochem. biophys. Res. Commun. 23, 195. StCgDER, W. R., SCnREmER, H. D. & SPENCER,J. N. (1973). Spectroehim. Acta 29A, 1225. Tn~'WALT,U. & BUt~G, C. E. (1972). J. Am. chem. Soc. 94, 8892. VAN Dtan,r~VELDT,F. B. (1969). In Intermotecular Forces and the Hydrogen Bond. Utrecht: Pressa Trajectina. WOL~an~G, M. & HFLMHOLZ,L. (1952). J. chem. Phys. 20, 837. ZACKmSSON,M. (1961). Acta chem. stand. 15, 1785.
Interactions of 6-thioguanine in B-DNA: Possible Mechanism of its Mutagenic Action H. CHOJNACKI AND W. A. SOKALSKI
Department of Physical Chemistry, Institute of Organic and Physical Chemistry, 50-370 Wroclaw, Wybrzef:e Wyspianskiego 27, Poland (Received 4 April 1974, and in revisedform 10 October 1974) Perturbation energies calculated for complementary base systems containing 6-thioguanine have been compared with the relevant results for normal base pairs. It is concluded that a single 6-thioguanine incorporated into B-DNA results in the increase of horizontal and vertical interactions whereas the respective stacking interactions between the same neighbours tend to decrease. The mutagenic action of the 6-thioguanine molecule in the DNA is interpreted as inhibiting of transcription and replication processes.
1. Introduction
The mechanism of the antitumour activity of 6-thioguanine has not yet been completely established. However, it is thought that the inhibitory action of this compound is a result of its incorporation into DNA replacing the normal guanine component (LePage, 1960; Kaplan, Smith & Tomlin, 1961; LePage & Jones, 1961; LePage & Junga, 1963). Related to this, Mautner & Jaff6 (1961) suggested that 6-thioguanine is able to form rather unusually strong hydrogen bonds with cytosine leading to interference of the replication process of DNA. Furthermore, some evidence has been presented indicating that sulphur containing molecular systems may interact more strongly than their oxygen analogues (Flett, 1953; Zackrisson, 1961; Niedzielski, Drago & Middaugh, 1964; Krueger, 1970; Snyder, Schreiber & Spencer, 1973). On the other hand, intermoleculer N - - H - . . S hydrogen bonds are considered to be weaker than the usually shorter bonds of the N - - H - - . O type (Donohue, 1969; Lutsky & Goncharova, 1972; Geller, Pohorille & Jaworski, 1973). Thus, the experimentally determined cases of stronger interactions of sulphur complementary base analogues are explained by increased vertical interactions (Scheit & Faerber, 1971; Faerber, Scheit & Sommer, 1972). Recent studies based on the comparative X-ray studies of 6-thioguanine, 6-thioguanosine and guanine (Bugg & Thewalt, 1970; t67
168
H. CHOJNACKI
AND W.
A. S O K A L S K I
Thewalt & Bugg, 1972) concluded that antineoplastic activity of 6-thioguanine was mainly due to alterations of hydrogen bond lengths of the respective base pairs, a study which seems to indicate that vertical interactions play a minor role. The controversial views mentioned above were an encouragement to compare theoretical interaction energies for 6-thioguanine and guanine base pairs of the B-DNA system.
2. Outline of the Calculation Method
The relative interaction energies have been calculated within the framework of the Rayleigh-Schr6dinger perturbation theory with the use of semiempirical parametrization of the LCMO method (Chojnacki & Sokalski, 1973). According to this approach the intermolecular interaction energy is given by E = E I + E II (1) where E ~ and E n are the first and second order perturbation energies, respectively. When intermolecular exchange effects are neglected the first order perturbation energy may be approximated to
= E Z (P,e,r,,- Pr Y
-- e, V,b + ZoZ /Ro )
S
representing an electrostatic term (Fujita, Imamura & Nagata, 1974; Fukui & Fujimoto, 1968). Neglecting dispersion and polarization forces the second order interaction energy becomes
ff ..o .f g} (z ~i
~"
(31 i
j.
E°-E~
resembling the charge-transfer (delocalization) contribution in the total interaction energy (Fukui & Fujimoto, 1968). The indices r and s denote atomic orbitals, a and b correspond to atomic centers, whilst the i and j are molecular orbitals belonging to the interacting molecules R and S, respectively at the R,b interatomic distance. Eigenvalues Ek, eigenvectors cut and electron populations P~ were evaluated by the use of the standard CNDO/2 approximation (Pople & Segal, 1966). The two centre Coulomb integrals F,~ and nuclear attraction integrals V,o and V,b were calculated with the ns Slater atomic orbitals. The resonance integrals H,s were approximated by the Wolfsberg & Helmholz (1952) formula H,s = 0.5K(H,, + Hs.~)S,. ~ (4)
INTERACTIONS OF 6-THIOGUANINE IN B-DNA
169
with K = 1 and the respective valence state ionization energies Hkk assumed according to Hinze & Jaff6 (1962) or in the case of a sulphur atom, taken from Levison & Perkins (1969). Overlap integrals S,. s were evaluated with the Slater basis set. The d atomic orbitals of sulphur were not considered explicitly as they do not seem to confer any significant contribution to the interaction energy (Guimon, Gonbeau & Pfister-Guillouzo, 1973). The method used permits a partitioning of the E n energy into tr--tr and re--re contributions. However, the partitioning is precise only in the case of coplanar systems since the overlap integrals were not separated into tr--o" and ~--rc components.
(-722) + 260
~-,53) -n
+~ ~
\~,,9' I
+~
~-"
/
r
f : ~ (- 646)
" -30
%.
\
/
+43
6
" ~ - -...~
II
#
(- 3
(~
rE) . . . . 3-3o~
~
(-624) |
+133
1
+6
+'~
\
/ ~
+,6
(--347) / /
\_
(+!87) +175
~
+ 6
....
( + 2 2 _ 2 ) ~ • 191 ~ J ~
1
- / ';
-'
./
+~"., (- 640) +471
+,4o
A -I- 135 H{~,)~l- 149
F1G. 1. Molecular geometry assumed with the respective charge distribution (milliproton units) of the 6-thioguanine-cytosine base pair calculated for isolated molecules by CNDO/2 method. The non-bracketted values are a-electron charges and those in parentheses re-electroncharges, respectively.
The geometry of the 6-thioguanine-cytosine system (Fig. 1) assumed in the calculation resembled that of the guanine-cytosine base pair of the WatsonCrick configuration. The N - H . • • S and C = S bond lengths were taken equal to 3.30 • and 1.78/~ respectively, instead of 3.00/~ and 1.20/~ for the normal guanine-cytosine base pair (Blizzard & Santry, 1969). The interplanar distance between neighbouring purines was assumed to be 3.38 A with the relative rotation of the components by 36 ° around the helix axis (Fig. 2) in accordance with the X-ray diffraction data for B-DNA (Arnott, Dover & Wonacott, 1969).
170
H. CHOJNACKI A N D W . A . SOKALSKI
FIG.2. Base stacking patterns in the B-DNA crystal fibres (Arnott et at., 1969) as viewed perpendic~ilarto the plane of the lower base represented by dashed lines. The cross is centred on the position of the helix axis.
3. Results and Discussion
The interaction energies calculated for 6-thioguanine-cytosine, guanineguanine, 6-thioguanine-guanine and 6-thioguanine-6-thioguanine systems partitioned into components are given in Tabb 1. In accordance with experimental information (Shoup, Miles & Becker, 1966; Kyogoku, Lord & Rich, 1969; Scheffler & Sturtevant, 1969; Pohl, 1973) and earlier theoretical calculations (Pullman & Pullman, 1968; Bertran, 1972; Danilov, Zheltovsky & Kudritskaja, 1973; Nagata, Imamura & Fujita, 1973; Fujita, TABLE 1 Partitioning of intermolecular interaction energies (kca1Jmol) for complementary brrses and their 6-thioguanine analogue Molecular system Adeninethyminet Guanine-cytosinet 6-thioguaqine-cytosinc
t Chojnacki & Sokalski (1973).
-5.95 -12.78 - 17.25
-13.25 -13.74 -18.76
-04010 -0.0007 -0~0011
-
-19.20 -26-52 -36.01
I N T E R A C T I O N S OF 6 - T H I O G U A N I N E IN B - D N A
171
Imamura & Nagata, 1974; Rein, 1974) the calculated interaction energies for guanine--cytosine are greater than those of the adenine-thymine system. The observed enthalpy changes for base pairs formation at physiological conditions are -7.90 + 0.14 kcal/mole for dA.dT base pair (Schettler & Sturtevant, 1969) and -11.1 + 0.5 kcal/mole for dG.dC base pair ~ohl, 1973). In our theoretical approach, neglecting exchange, dispersion and polarization terms seems to be a rather drastic assumption. The role of neglected terms for small hydrogen bonded systems has been pointed out recently (Kollman & Allen, 1972; Daudey, 1974). However, at the equilibrium geometry of different molecular pairs the repulsive exchange contribution and the attractive dispersion and polarization terms tend to cancel each other (or tend to be constant) leaving the relative ordering of stabilization energies to be determined by the Coulomb and charge transfer interactions (Person, 1973). Hence, the limited success of simple electrostatic or charge transfer theories used in the past for description of small hydrogen bonded systems and typical charge transfer complexes respectively, may be justified. For large hydrogen bonded systems the charge transfer interactions seem to be more important (Malarski, 1974). On the other hand the charge transfer stabilization energy allows one to obtain a good correlation with observed heat of formation for tetracyanoethylene molecular complexes with various aromatic hydrocarbons (Herndon & Feuer, 1968). Therefore, we decided to use the sum of both contributions as a relative intermolecular energy for similar large molecular systems at equilibrium distances. However, the quantities calculated here in any case cannot be considered as absolute values of stabilization energy. The most advanced studies done hitherto within the scheme of perturbation theory do not reproduce properly potential energy curves even for small hydrogen bonded systems (Van Duijneveldt, 1969; Daudey, 1974). It is because of great mathematical complications in solving the intermolecular interaction problem reviewed recently by Certain & Bruch (1972). It is dit~cult to estimate the role of the solvent in the interaction energy as well as possible changes in the electronic structure of the complementary bases caused by neglecting the sugar moieties. The sulphur orbitals were assumed to have the same hybridization as the oxygen atom. However, the density maps obtained for the thiophene molecule indicate that atomic orbitals on sulphur are, in comparison with oxygen in the furan molecule, only slightly hybridized (M~trtensson & Chojnacki, 1973). Thus, the lone pair of sulphur is more diffuse than the oxygen atom lone pair resulting in a decrease of the overlap integral between sulphur and the hydrogen atom in the hydrogen bond. As seen from Table 1, the changes in the horizontal
172
H.
CHOJNACKI
AND
W.
A. S O K A L S K I
interaction energy upon replacing oxygen by sulphur atom in the systems looked at, are mainly due to charge transfer interactions as was found from IR measurements of similar systems (Malarski, 1974). It is concluded that the relative strengths of hydrogen bonds of the N - H - " • S and N - H . • • O type depend on the molecular geometry and electronic structure of interacting systems and must be considered individually. Assuming that, in spite of approximations made in the calculation, the qualitative relation between theoretical interaction energies still gives the higher horizontal interaction energy for the 6-thioguanlne-cytosine pair than that of the normal guanine-cytosine pair. Thus, 6-thioguanine incorporated into DNA may exert an inhibiting action on replication and transcription processes associated with the respective breaking of intermolecular hydrogen bonds. The results obtained indicate the importance of vertical interactions as well. In this case dispersion and polarization forces may contribute a greater share to the stabilization energy (Bertran, 1972; Danilov et al., 1973). The stacking interaction energies ordered as 6-thioguanineguanine > guanine-guanine > 6-ttfioguanine-6-thioguanlne indicate that vertical interactions for single molecules of 6-thioguanine incorporated into DNA should increase in comparison to the pertinent interactions of guanine. The destabilizing stacking interactions found for two adjacent 6-thioguanine molecules may lead to a distortion of the secondary structure of D N A at the incorporation site. In the single step of the replication process, a deficiency of energy at the region of replicase enzyme may appear. It would be the case when the energy gained from hydrolysis of nucleosidetriphosphate together with that of formation of hydrogen bonds and stacking interactions in the region of the new formed polynucleotide chain is less than the energy needed to cleavage the stronger hydrogen bonds in 6-thioguanine-cytosine pair together with the binding energy nucleosidemonophosphate to polynucleotide chain. Such a situation is more probable in transcription than replication where in the single step of the process, breaking of the two hydrogen bonds is involved. Other support of the supposition comes from the observation that the inhibitory action of 6-thioguanine is more pronounced in the RNA than in the DNA synthesis (Mandel, Latimer & Riis, 1965). It does not mean, however, that the incorporation of 6-thioguanine into nucleic acid has in every case led to lethal mutation since incorporation process into R N A has been completely inhibited only in the presence of blocking transcription actinomycin D (Kwan, Kwan & Mandel, 1973). It is concluded that mutagenic effects of 6-thioguanine may be mainly due to its incorporation into DNA although its incorporation into various fractions of R N A has been determined also (Gray & Rachmeler, 1967; Kwan et al., 1973). The confirmation of the above
INTERACTIONS OF 6 - T H I O G U A N I N E IN B-DNA
173
hypothesis derives f r o m studies o n t u m o u r s susceptible o r resistant t o the 6-thioguanine action. I t was e s t a b l i s h e d t h a t in susceptible t u m o u r s , 6-thioguanine is i n c o r p o r a t e d m a i n l y into D N A a n d into R N A in resistant lines (LePage & Junga, 1963). The numerical computation performed in part by Professor O. M ~ t e n s s o n at the Quantum Chemistry G r o u p o f the Uppsala University is warmly acknowledged. This work has been sponsored in part by the Polish Academy of Sciences under the P A N - 3 contract. REFERENCES ARNOYr, S., DOVER, S. D. & WONACOrr, A. J. (1969). Aeta crystallogr. B25, 2192. BERTRAN,J. (1972). J. theor. Biol. 34, 353. BLIZZARD,A. C. & SAr~TRY,D. P. (1969). J. theor. BioL 25, 461. BUGG, C. E. & THEWALr, U. (1970). J. Am. chem. Soc. 92, 7441. CERTaiN, P. R. & BRUCH, L. W. (1972). M T P lnt. Bev. Sci. Phys. Chem. 1, 113. C-'nOJNACKr,H. & SOKALSrd,W. A. (1973). J. molec. Struct. 15, 263. DANILOV,V. I., ZHEL'rOVSKY,N. V. & KODmrSr,~JA, Z. G. (1973). Preprint ITP-73-109R, Kiev. DAUDEY, J. P. (1974). Int. J. Quantum Chem. 8, 29. DONOrlUE, J. (1969). J. molec. Biol. 45, 231. FAERBER,P., SCHEIT,K. H. & SOMMER,H. (1972). Eur. J. Biochem. 27, 109. FLEa'r, M. S. C. (1953). J. chem. Soc. 347. FUJ1TA,H., IMAMURA,A. & NACArA, C. (1974). J. theor. BioL 45, 411. FUKUI, K. & FUJIMOTO,H. (1968). Bull. chem. Soc. Japan 41, 1989. GELLER, M., POHORILLE,A. & JAWORSKI,A. (1973). Biochim. biophys. Acta 331, 1. GRAY, P. N. & RACHMELER,M. (1967). Biochhn. biophys. Aeta 138, 432. GUIMON, C., GOr,rBEAU,D. & PFXSTER-GUILLOUZO,G. (1973). J. molec. Struet. 16, 271. HERNDON, W. C. & FEUtR, J. (1968). J. Am. chem. Soe. 90, 5914. I-IIr~ZE,J. & JAFrd, H. H. (1962). J. Am. chem. Soc. 84, 540. KAPLAN,H. S., SMrrH, D. C. & TOMUN, P. (1961). Nature, Lond. 190, 794. KOLLMAN,P. A. & ALLEN, L. C. (1"972). Chem. Rev. 72, 283. KRUEGER, P. J. (1970). Tetrahedron 26, 4753. KWAN, S. W., KWAN, S. P. & MANDEL,H. G. (1973). Cancer Res. 33, 950. KYO~OKU, Y., LORD, R. C. & RICH, A. (1969). Bioehim. biophys. Acta 179, 10. LEPAC;E,G. A. (1960). Cancer Res. 20, 403. LEPAGE, G. A. & JON~, M. (1961). Cancer Res. 21, 1590. LEPAGE, G. A. & JUNGA,I. G. (1963). Cancer Res. 23, 739. LEVISlON,K. A. & PERKINS,P. G. (1969). Theor. chim. Acta 14, 206. LUTSKY,A. E. & GONCHAROVA,E. I. (1972). Ukr. khim. Zh. 38, 1223. MALARS~, Z. (1974). Roczn. Chem. 48, 663. MANDEt., H. G., LA~MER, R. G. & PallS, M. (1965). Biochem. Pharmac. 14, 661. M~,RT~NSSON,O. & CHOJrqACKI,H. (1973). Actaphys. poL A44, 259. MAUTNER, H. G. & JAFFa, J. J. (1961). Biochem. Pharmac. 5, 343. NAGATA,C., IMAMURA,A. & FUJITA, H. (1973). Adv. Biophys. 4, 1. NmDZmLSKI, R. J., DRACO, R. S. & MIDDAUOH, R. L. (1964). J. Am.chem. Soc. 86, 1964. PERSON, W. B. (1973). In Spectroscopy and Structure of Molecular Complexes (J. Yarwood, ed.) p. 15. London: Plenum Press. POHL, F. M. (1973). Eur. Jr. Biochem. 42, 495. POPLE, J. A. & SEOAL,G. A. (1966). J. chem. Phys. 44, 3289. POLLMAN,A. & PULLMAN,B. (1968). Adv. Quant. Chem. 4, 267.
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