Molecular Modeling of Nucleic Acids - American Chemical Society

Chapter 5

Hydrogen-Bonding Patterns Observed inthe Base Pairs of Duplex Oligonucleotides 1,4

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William N. Hunter , Gordon A. Leonard , and Tom Brown Downloaded by GEORGE MASON UNIV on March 30, 2016 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch005

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Department of Biochemistry, University of Dundee, Dundee DD1 4HN, Scotland European Synchrotron Radiation Facility, F-38043 Grenoble, Cedex, France Department of Chemistry, University of Southampton, Highfield, Soughampton SO17-1BJ, United Kingdom 2

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Watson-Crick base pairing in duplex structures utilises a unique combination of hydrogen bond donors and acceptors to stabilise the association. This stability contributes to the fidelity of replication. Non -Watson-Crick base pairs, mismatches or base pairs involving chemically modified bases adopt different patterns of hydrogen bond interactions sometimes utilising water molecules to stabilise the pairing and in some cases an argument can be put forward forC - H • • • Ointeractions as a contributing factor in the pairing and not just in mismatches. The different hydrogen bonding patterns deduced on the basis of single crystal x-ray diffraction studies are described. These hydrogen bonding patterns have implications for protein-nucleic acid interactions and for the modelling of nucleotide structures. Single crystal x-ray diffraction methods have been used to characterise numerous oligonucleotide structures enabling studies of the fine structure of D N A , oligonucleotide hydration, interactions with small molecule ligands and proteins. A particular focus has been on non-standard base associations and researchers have sought to characterise different non-Watson-Crick base pairs to further understanding of their influence on duplex D N A and RNA, and to investigate which structural features might be used in recognition and repair of these errors in DNA. Bases that can be chemically modified present distinct hydrogen bonding patterns and these too have been investigated. In this article we re-examine the hydrogen bonding patterns of the Watson-Crick pairing and survey some of the non-Watson-Crick base associations in duplex D N A and RNA. Complementarity of the Watson-Crick pairs. Watson and Crick recognised that a complementary base pairing scheme in duplex D N A contributed to the fidelity of replication ( 1 ). Purines interact with pyrimidines so that G pairs with C and A pairs with Τ to form what are termed Watson-Crick base pairs (Fig. 1). The very specific manner in which the Watson-Crick base pairs are formed contributes stability to an oligonucleotide structure and a particular arrangement of functional groups to interact with enzymes and proteins using specific hydrogen bonding patterns (2). Since the human genome is estimated to contain around 10^ base pairs it is not surprising that Corresponding author © 1998 American Chemical Society

Leontis and SantaLucia; Molecular Modeling of Nucleic Acids ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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MOLECULAR MODELING OF NUCLEIC ACIDS

Downloaded by GEORGE MASON UNIV on March 30, 2016 | http://pubs.acs.org Publication Date: November 26, 1997 | doi: 10.1021/bk-1998-0682.ch005

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5. HUNTER ET AL.

Hydrogen Bonding in Duplex Oligonucleotides

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mistakes occur during replication. A single error in a triplet can be carried through and eventually lead to a serious mutation in the gene product. Errors may be introduced via non-Watson-Crick base pairs termed mismatches or mispairs. Alternatively, damage to D N A can produce bases with altered chemical properties capable of scrambling the genetic code (3). Mistakes, if unchecked, can be deleterious and a complex protein recognition and repair system makes a contribution to maintaining the fidelity of replication (4). Studies on these systems represent an exciting area of structural biology (5). Crystallographic studies of mismatches and modified bases in D N A and R N A complement thermodynamic studies on stability of the mismatches or base pairs involving chemically modified components (6). Our objective in this article is to reconsider the conventional view of hydrogen bonding in Watson-Crick base pairs and to describe some of the different hydrogen bonding patterns observed in non-WatsonCrick base associations in duplex structures. There are additional base-associations in larger R N A fragments, for example tRNA, which we will not cover and readers are directed elsewhere for coverage of those structures (2, 7). A Definition for the Hydrogen Bond. Before a description of the varied base pair hydrogen bonding patterns that have been observed in oligonucleotide structures is provided, it is instructive to consider what constitutes a hydrogen bond. Initially, it was thought that a hydrogen bond could only be formed if the donor-atom-acceptor atom distance was less than the sum of their van der Waals radii (8). So, based on distances observed from single crystal X-ray diffraction studies hydrogen bonding could be assigned. It was later realised that this distance restraint was too strict and ignored the electrostatic arguments that the attractive energy diminishes linearly with increasing distance and so the assignment of hydrogen bonds should not be confined by such overly strict distance criteria (9). The most appropriate definition, in our opinion, has been provided by Steiner and Saenger (10). They defined a hydrogen bond as "any cohesive interaction Χ-Η···Υ, where Η carries a positive charge and Y a negative (partial or full) charge, and the charge on X is more negative than on H". Such a définition is attractive and can be widely applied to accurate X-ray structures or to less accurate modelling excercises (11). The conventional hydrogen bonds formed by the nucleotide bases involve N Η · · · 0 and Ν-Η···Ν interactions. The existence of C - H O hydrogen bonds has in the past been a source of controversy yet now the role of this type of interaction in stabilising molecular assemblies is widely recognised (12,13). Consideration of bond polarization effects suggests that some C - H « " 0 interactions satisfy the definition of the hydrogen bond as given above. w

Watson-Crick pairing. The Α·Τ and G»C pairs are shown in Figure 1. The conventional description of the hydrogen bonding is that the G*C is held together by three Η-bonds whilst the Α·Τ is held together by only two Η-bonds. We had previously analysed the detailed geomety of the Α·Τ pairing and suggested that there is in fact a third Η-bond, a C - H " » 0 interaction on the minor groove side (14). Recent theoretical studies have supported this idea (15). This is not a strong stabilising interaction but rather it serves the purpose of alleviating the destabilising effects of having an unfulfilled hydrogen bond donor or acceptor group in the structure. The incorporation of such a weak interaction may be a useful consideration in molecular modelling of nucleic acids. Mismatches. Watson-Crick Α·Τ or G*C pairs have to compete with 8 non-WatsonCrick alternatives termed mispairs or mismatches. These are the purine-pyrimidine ϋ · Τ and A O pairings, the purine-purine G < J , Α·Α and G»A and the pyrimidinepyrimidine C»C, Τ·Τ and C»T mismatches. Mutagenic pathways are divided into



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transition and transversion paths. The former invokes purine-pyrimidine mismatches, the latter purine-purine or pyrimidine-pyrimidine mispairs. The incorporation of non-Watson-Crick base pairs in duplex D N A is the most common error occurring on replication. The theory of mispair formation was initially proposed by Watson and Crick (16, (extended by Topal and Fresco (17) and extensively reviewed by Strazewski and Tamm (18)) and postulated the involvement of rare tautomer forms of the bases. The mismatches involving these tautomers would be sterically equivalent to Watson-Crick base pairs and unlikely to distort the duplex into which they are formed. The crystallographic study of mispairs cannot give any information on the occurrence of rare tautomers during the replication process. The resolution to which most structures are determined does not allow for the precise location of hydrogen atoms and these have to be inferred using geometric considerations. One of the main conclusions from mismatch studies is that there is no need to invoke the presence of rare tautomers in mismatch formation and stability. Crystallographic study of mismatches have concentrated on sequences known to crystallise into which the mispairs were engineered. A common framework has been the B - D N A Drew-Dickerson dodecamer duplex, d(CGCGAATTCGCG) (19). Other templates have been Α-form D N A octamers and Z-form hexamers (20). In each case a duplex containing two mispairs has been formed. This approach maximises the chances of getting well ordered single crystals and means that there is a native WatsonCrick structure for comparative purposes. Purine-pyrimidine base pairs. The G*T pair was the first mismatch to be characterised and this in an Α-form octamer (21, 22). The mispair was subsequently studied in different sequence environments and in different D N A forms (23-25). This purine-pyrimidine pair adopts what is termed the wobble configuration that was first proposed by Crick to explain G*U pairing at the third codon position during codonanticodon interactions (26). In this mispair, the purine is shifted towards the D N A minor groove, the pyrimidine towards the major groove. The bases maintain the major tautomeric forms and create two inter-base hydrogen bonds (Fig. 2a). Solvent molecules bridge functional groups on the bases in both grooves and confer added stability. In addition, G U ^ and G U ^ pairs (where uracil contains a bromine or fluorine at the 5 position) have been characterised in Z-form hexamers (27, 28) and wobble G*U pairs plus attendant solvent molecules observed in a fragment of 5S rRNA (29). Inosine (I) is a guanine analogue lacking the 2-amino group. It is commonly found in tRNA and is able to pair with A, C and U in codon-anticodon interactions. This is an important base since the ability to pair with three other bases contributes to the degeneracy of the genetic code. Inosine occurs rarely in D N A , as a result of deamination of deoxyguanosine, where it is potentially mutagenic. The Ι·Τ pair (30) assumes a similar structure to the G*T although the loss of N2 on the minor groove side of the duplex removes the possibility of a stabilising water bridge between the bases. The A«C mismatch is similar to the G*T, but there are two arrangements that need to be considered to explain the formation of two Η-bonds Unking the bases (31, 32), (Fig 2b, c). A solvent molecule can link the bases on the major groove side to aid stability but not on the minor groove side. The adenine is either protonated or in a rare tautomeric form. Energetic considerations support the former and biophysical characterisation of A»C mispairs using N M R and U V melting methods over a wide pH range were subsequently to prove this (33). This pair should be denoted as A C . e

r

e

+ e

Purine-Purine Base Pairs. A*G and G*G pairs have both been characterised in duplex B-DNA. The Α·Α pairing has only been observed in R N A structures (34, 7



5. HUNTER ET AL.


Ν—H / H

G.Τ

OR

A+.C

A(imino).C Figure 2. (a) The wobble ϋ · Τ (b) the Α+·0 and (c) A«C with the adenine in the imino form.


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and references theirin). Biochemical studies have indicated that A*G mismatches are repaired with much less efficiency than other mispairs (35). Structural studies have identified four G»A configurations in D N A (36-39), (Fig. 3). The form of the mispair that is observed is dependent on the pH, salt concentration and in particular on the sequence environment. The sequence dependence of the G A conformation can be rationalised by dipole-dipole interactions with adjacent bases (40). The possibility of forming a hydrogen bond using a functional group provided by an adjacent base can also be important. This is clear in the example of the G(anti)*A(anti) pairing where the presence of an inter-base-pair hydrogen bond with the amino N2 of guanine to the 02 of an adjacent thymine on the opposing strand has been noted (36). Without an 02 in this position some other G*A conformation might be preferred. The key point about studies on the GrA mispair is that the variablility of conformations that can be observed would present quite a challenge to an enzyme recognition and repair system and this may be the reason for low levels of G*A mismatch repair. In the R N A duplex structure, r ( C G C G A A U U A G C G ) there are two A(anti) G(anti) base pairs and evidence to suggest the same degree of variability observed in D N A (38). A careful investigation of the hydrogen bonding possibilities indicates that the A(anti)*G(anti) pairing uses a conventional hydrogen bond formed between N6 and 0 6 and what is termed a reverse, three-center hydrogen bond in which the lone pair on N l is shared with the N - H groups of the guanine N l and N2. This avoids the destabilising effects of having unsatisfied hydrogen bonding functional groups. The structural variation of the G A mismatch also applies to Ι·Α pairs (40-42) and may help explain the mutagenicity of inosine. U V melting studies suggest that inosine containing mismatches are quite stable (43) whereas most other mispairs destabilise the D N A duplex and produce local melting effects that promote strand dissociation. Repair enzymes may utilise such a physical property of the mismatch duplex to recognise incorrect base pairing. Local déstabilisation would also assist the flipping out of mismatched bases for excision. The phenomenon of base flipping as part of the protein recognition and repair process has been noted on the basis of crystallographic studies (5). There has only been one duplex structure characterised which contains two homopurine GC mispair observed in an R N A fragment. W represents the water molecule that bridges the bases.


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86 Br

r ( G C U U C G G C ) d ( U ) has a similar U - U pair at the end of one of the helices which is disordered (48) The hexanucleotide, r(UUCGCG) crystallises with a tetranucleotide duplex involving O G pairs and two U U pairs formed by the overhanging bases of neighbouring duplexes (49). There is a conventional hydrogen bond between N3 to 04 but also a C-H**0 hydrogen bond between C5 and 04 (Fig. 6b).


e

Pairings with Modified Bases. To compound the pressures of carrying out the replication of D N A involving many bases, the genetic code is constantly under attack from chemical and physical forces in the environment or that are generated in cells during the normal course of metabolism. Carcinogenic chemicals, ultraviolet light, ionising radiation and reactive oxygen species can all produce modifications to D N A (3,4). Of particular interest are alterations to the purines and a number of examples are depicted in Figure 7. Guanine reacts with alkylnitrosoureas to form 0-6-methylguanine (06MeG) which is potentially very damaging since it alters the hydrogen bonding potential of the base. The effect can be to promote G to A transition mutations. The 06MeG«T mispair could be selected during replication in preference to a 06MeG«C pair. The crystal structure of a 06MeG»C pair has been determined at physiological pH (50-52) and is shown to adopt a wobble conformation (Fig. 7a). Chemical damage is not only induced by alkylating agents. Vinyl chloride, for example, reacts with adenine producing 1, N -ethenoadenosine (edA). The structure of the G»edA pairing has been determined (53) and the association is depicted in Figure 7d. There are two obvious hydrogen bonds and a C - H O hydrogen bond has been invoked between the 8H and 06 of G to alleviate the déstabilisation of an unsatisfied hydrogen bond acceptor in the pair. Unlike many of the other nonWatson-Crick pairings that have been characterised, the G»edA pairing produces a significant distortion of the sugar-phosphate backbone. Alterations in the bond angles associated with the furanose-phosphate backbone lead to a bulge in the structure. Such perturbation might represent a signal for the recognition and repair of this modified base by 3-methyladenine-DNA glycosylase. Purines undergo oxidation at the 8-position to produce 8-oxoadenine (08A) and 8-oxoguanine (08G) where the bases are predominantly in the keto form. Modification at the 8-position does not affect the hydrogen bonding patterns in G»C and Α · Τ pairs but the presence of the 8-0 and N 7 H does promote alternative possibilities and a syn conformation about the glycosidic bond. This has been observed in structures of 0 8 G - A and 0 8 A - G pairings (54,55 Fig 7e, 7f). The highly mutagenic 0 8 G in genomic D N A can facilitate the G to Τ transversion mutation via an intermediate 08G*A base pair. Such a pairing has been shown to be fairly stable. This property in combination with a psuedosymmetry about the glycosidic bonds suggests why this pair is not readily recognised by proof reading enzymes. 0 8 A is not very mutagenic and the 08A*G pairing whilst again showing a syn anti pair is asymmetric about the glycosidic bonds, a structural feature that may assist recognition and repair. Four bifurcated hydrogen bonds resulting from two reverse three centred hydrogen bonding systems hold the bases in place. A l l functional groups participate in hydrogen bonds. 6

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5. HUNTER ETAL.


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a

G(6)-MeG.C

b

H

0(6)-MeG.C+

c

H

0(6)-MeG.T e

Figure 7. (a) The 06 - MeG»C pair which mimics the G T mismatch, (b) The 06 MeG»C pairing which resembles a Watson - Crick base pair, (c) 06 - M e G T mismatch which also resembles a Watson - Crick pair, (d) The G (anti)*edA pair where edA is ethenoA. (e) The A(anti)*0%G(syn) and (f) G(antî)*0%A(syn) pairings where 0 8 G and 0 8 A represent 8 - oxoG and 8 - oxoA respectively. +

e

Continued on next page Leontis and SantaLucia; Molecular Modeling of Nucleic Acids ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


G(anti).8-oxoA(syn) Figure 7. Continued.


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Acknowledgments. The Wellcome Trust, the Biotechnology and Biochemistry Science Research Council, the Engineering and Physical Sciences Research Council have provided support for our studies. Dr T. Steiner is thanked for permission to reference unpublished material and for sharing his results with us.


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Leonard, G.A. (1989) Nucleic Acids Res. 17, 55-72. (31) Hunter, W.N., Brown, T., Anand, N.N. and Kennard, O. (1986) Nature 320, 552-555. (32) Hunter, W.N., Brown, T. and Kennard, O. (1987) Nucleic Acids Res. 15, 6589-6606. (33) Brown, T., Leonard, G.A., Booth, E.D. and Kneale, G. (1990)J. Mol. Biol. 221, 437-440. (34) Baeyens, K.J., De Bondt, H.L., Pardi, A. and Holbrook, S.R. (1996) Proc. Natl. Acad. Sci. USA. 93, 12851-12855. (35) Fersht, A.R., Knill-Jones, J.W. and Tsui, W.C. (1982) J. Mol. Biol. 156, 3751. (36) Prive, G.G., Heinemann, U., Kan, L.S., Chandrasegaran, S., and Dickerson, R.E. (1987) Science 238, 498-504. (37) Brown, T., Hunter, W.N., Kneale, G.G., and Kennard, O. (1986) Proc. Natl. Acad. Sci. USA 83, 2402-2406. (38) Brown, T., Leonard, G.A., Booth, E.D. and Chambers, J. (1989) J. Mol. Biol. 207, 455-457. (39) W.N. Hunter, T. Brown and O. Kennard. (1986) J. Biomolecular Structure and Dynamics. 4, 173-191. (40) Leonard, G.A., McAuley-Hecht, K., Abel, S., Lough, D.M., Brown, T. and W.N Hunter, W.N. (1994) Structure 2, 483-494. (41) Corfield, P.W.R., Hunter, W.N., Brown, T., Robinson, Ρ and Kennard, Ο (1987) Nucleic Acids Res. 15, 7935-7949. (42) Webster, G.D., Sanderson, M.R., Skelly, J.V., Neidle, S., Swann, P.F., Li, B.F. and Tickle, I. (1990) Proc. Natl. Acad. Sci. USA. 87, 6693-6697. (43) G.Leonard, E.Booth, W.N. Hunter, T. Brown. (1992) Nucleic Acids Res. 20, 4753-4759. (44) Skelly, J.V., Edwards, K.J., Jenkins, T.C. and Neidle, S. (1993) Proc. Natl. Acad. Sci. USA. 90, 804-808. (45) Holbrook, S.R., Cheong, C., Tinoco Jr, I. and Kim, S. H. (1991) Nature 353, 579-581. (46) Baeyens, K.J., De Bondt, H.L. and Holbrook, S.R. (1995) Nature Structural Biology 2, 56-62. (47) Lietzke, S.E., Barne, C.L., Bergland, J.A., and Kundrot, C.E. (1996) Structure 4, 917-930. (48) Cruse, W.B.T., Saludjian, P., Biala, E., Strazewski, P., Prange, T. & Kennard, O. Proc. Natl. Acad. Sci. (1994) 91, 4160-4164. (49) Wahl, M.C. Rao, S.T. and Sundaralingham, M. (1996) Nature Structural Biology 3, 24-30. (50) Leonard, G.A., Thomson, J.B., Watson, W.P. and Brown, T. (1990) Proc. Natl. Acad. Sci. USA 87, 9573-9576. (57) Ginell, S.L., Vojtechovsky, J., Gaffney, B., Jones, R. and Berman, H.M. (1994) Biochemistry 33, 3487-3493. (52) Vojtechovsky, J., Eaton, M.D., Gaffney, B., Jones, R., Berman, H.M. (1994) Biochemistry 34, 16632-16640. (53) Leonard, G.A., McAuley-Hecht, K.E., Gibson, N.J., Brown, T., Watson, W.P. and Hunter, W.N. (1994) Biochemistry 33, 4755-4761. (54) Leonard, G.A., Guy, Α., Brown, T., Teoule, R. and Hunter, W.N. (1992) Biochemistry 31, 8415-8420. (55) McAuley-Hecht, K.E., Leonard, G.A., Gibson, N.J., Thomson, J.B., Watson, W.P., Hunter, W.N. and Brown, T. (1994) Biochemistry 33, 10266-10270.


Molecular Modeling of Nucleic Acids - American Chemical Society

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