Refined crystal structure of an octanucleotide duplex with G . T mismatched base-pairs

Single crystal X-ray diffraction techniques have been used to determine the structure of the DNA octamer d(G-G-G-G-C-T-C-C) at a resolution of 2.25 A. The asymmetric unit consists of two strands coiled about each other to produce an A-type DNA helix. The double helix contains six G . C Watson-Crick base-pairs and two G . T mismatched base-pairs. The mismatches adopt a "wobble" type structure in which both bases retain their major tautomer forms. The double helix is able to accommodate this G . T pairing with little distortion of the overall helical conformation. Crystals of this octamer melt at a substantially lower temperature than do those of a related octamer also containing two G . T base-pairs. We attribute this destabilization to disruption of the hydration network around the mismatch site combined with changes in intermolecular packing. Full details are given of conformational parameters, base stacking, intermolecular contacts and hydration involving 52 solvent molecules.     

The structure of guanosine-thymidine mismatches in B-DNA at 2.5-A resolution

The structure of the deoxyoligomer d(C-G-C-G-A-A-T-T-T-G-C-G) was determined at 2.5-A resolution by single crystal x-ray diffraction techniques. The final R factor is 18% with the location of 71 water molecules. The oligomer crystallizes in a B-DNA-type conformation, with two strands interacting to form a dodecamer duplex. The double helix consists of four A X T and six G X C Watson-Crick base pairs and two G X T mismatches. The G X T pairs adopt a "wobble" structure with the thymine projecting into the major groove and the guanine into the minor groove. The mispairs are accommodated in the normal double helix by small adjustments in the conformation of the sugar phosphate backbone. A comparison with the isomorphous parent compound containing only Watson-Crick base pairs shows that any changes in the structure induced by the presence of G X T mispairs are highly localized. The global conformation of the duplex is conserved. The G X T mismatch has already been studied by x-ray techniques in A and Z helices where similar results were found. The geometry of the mispair is essentially identical in all structures so far examined, irrespective of the DNA conformation. The hydration is also similar with solvent molecules bridging the functional groups of the bases via hydrogen bonds. Hydration may be an important factor in stabilizing G X T mismatches. A characteristic of Watson-Crick paired A X T and G X C bases is the pseudo 2-fold symmetry axis in the plane of the base pairs. The G X T wobble base pair is pronouncedly asymmetric. This asymmetry, coupled with the disposition of functional groups in the major and minor grooves, provides a number of features which may contribute to the recognition of the mismatch by repair enzymes.     

Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Structural Studies of DNA Fragments: The G·T Wobble Base Pair in A,B and Z DNA; The G·A Base Pair in B-DNA

Abstract

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G·T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with 02 of T and 06 of G with N3 of T. The X-ray analyses establish that the G·T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G·A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.     

Structures of mismatched base pairs in DNA and their recognition by the Escherichia coli mismatch repair system.

The Escherichia coli mismatch repair system does not recognize and/or repair all mismatched base pairs with equal efficiency: whereas transition mismatches (G X T and A X C) are well repaired, the repair of some transversion mismatches (e.g. A X G or C X T) appears to depend on their position in heteroduplex DNA of phage lambda. Undecamers were synthesized and annealed to form heteroduplexes with a single base-pair mismatch in the centre and with the five base pairs flanking each side corresponding to either repaired or unrepaired heteroduplexes of lambda DNA. Nuclear magnetic resonance (n.m.r.) studies show that a G X A mismatch gives rise to an equilibrium between fully helical and a looped-out structure. In the unrepaired G X A mismatch duplex the latter predominates, while the helical structure is predominant in the case of repaired G X A and G X T mismatches. It appears that the E. coli mismatch repair enzymes recognize and repair intrahelical mismatched bases, but not the extrahelical bases in the looped-out structures.     

Base-base mismatches. Thermodynamics of double helix formation for dCA3XA3G + dCT3YT3G (X, Y = A,C,G,T).

Thermodynamic parameters for double strand formation have been measured for the sixteen double helices of the sequence dCA3XA3G.dCT3YT3G, with each of the bases A, C, G and T at the positions labelled X and Y. The results are analyzed in terms of nearest-neighbors and are compared with thermodynamic parameters for RNA secondary structure. At room temperature the sequence (Formula: see text) is more stable than (Formula: see text) and is similar in stability to (Formula: see text) and (Formula: see text) are least stable. At higher temperatures the sequences containing a G.C base pair become more stable than those containing only A.T. All molecules containing mismatches are destabilized with respect to those with only Watson-Crick pairing, but there is a wide range of destabilization. At room temperature the most stable mismatches are those containing guanine (G.T, G.G, G.A); the least stable contain cytosine (C.A, C.C). At higher temperatures pyrimidine-pyrimidine mismatches become the least stable.     

Single base mismatches in DNA. Long- and short-range structure probed by analysis of axis trajectory and local chemical reactivity

We have devised a procedure to generate any single base mismatch in a constant sequence context, and have studied these from two points of view. (1) We have examined electrophoretic mobility of 458 base-pair fragments containing approximately centrally located single mismatches, in polyacrylamide gels, compared to fully matched DNA fragments. We found that no single mismatch caused a significant perturbation of gel mobility, and we conclude that all the mismatches may be accommodated within a helical geometry such that there is no alteration of the path of the helix axis in a straight DNA molecule. (2) We have studied all the single mismatches with respect to reactivity to a number of chemical probes. We found that: (a) No mispaired adenine bases are reactive to diethyl pyrocarbonate and are therefore not simply unpaired such that N-7 is exposed. (b) A number of mispaired thymine bases are reactive to osmium tetroxide, and cytosine bases to hydroxylamine. (c) Where crystal or nuclear magnetic resonance structures are available, the reactivity correlates with exposure of the pyrimidine 5,6 double bonds to attack in the major groove as a result of wobble base-pair formation. This is particularly clear for G.T and I.T base-pairs. (d) Reactivity of bases in mismatched pairs can be dependent on sequence context. (e) Reactivity of the C.C mismatch to hydroxylamine is suppressed at low pH, suggesting that a rearrangement of base-pairing occurs on protonation. The results overall are consistent with the formation of stacked intrahelical base-pairs wherever possible, resulting in no global distortion of the DNA structure, but specific enhancement of chemical reactivity in some cases.     

G.T wobble base-pairing in Z-DNA at 1.0 A atomic resolution: the crystal structure of d(CGCGTG).

The DNA oligomer d(CGCGTG) crystallizes as a Z-DNA double helix containing two guanine-thymine base pair mismatches of the wobble type. The crystal diffracts to 1 A resolution and the structure has been solved and refined. At this resolution, a large amount of information is revealed about the organization of the water molecules in the lattice generally and more specifically around the wobble base pairs. By comparing this structure with the analogous high resolution structure of d(CGCGCG) we can visualize the structural changes as well as the reorganization of the solvent molecules associated with wobble base pairing. There is only a small distortion of the Z-DNA backbone resulting from introduction of the GT mismatched base pairs. The water molecules cluster around the wobble base pair taking up all of the hydrogen bonding capabilities of the bases due to wobble pairing. These bridging water molecules serve to stabilize the base-base interaction and, thus, may be generally important for base mispairing either in DNA or in RNA molecules.     

Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies.

We investigated the effects of various primer-template mismatches on DNA amplification of an HIV-1 gag region by the polymerase chain reaction (PCR). Single internal mismatches had no significant effect on PCR product yield while those at the 3'-terminal base had varied effects. A:G, G:A, and C:C mismatches reduced overall PCR product yield about 100-fold, A:A mismatches about 20-fold. All other 3'-terminal mismatches were efficiently amplified, although the G:G mismatches appeared to be more sensitive to sequence context and dNTP concentrations than other mismatches. It should be noted that mismatches of T with either G, C, or T had a minimal effect on PCR product yield. Double mismatches within the last four bases of a primer-template duplex where one of the mismatches is at the 3' terminal nucleotide, in general, reduced PCR product yield dramatically. The presence of a mismatched T at the 3'-terminus, however, allowed significant amplification even when coupled with an adjacent mismatch. Furthermore, even two mismatched Ts at the 3'-terminus allowed efficient amplification.     

Specific binding of human MSH2.MSH6 mismatch-repair protein heterodimers to DNA incorporating thymine- or uracil-containing UV light photoproducts opposite mismatched bases.

Previous studies have demonstrated recognition of DNA-containing UV light photoproducts by bacterial (Feng, W.-Y., Lee, E., and Hays, J. B. (1991) Genetics 129, 1007-1020) and human (Mu, D., Tursun, M., Duckett, D. R., Drummond, J. T., Modrich, P., and Sancar, A. (1997) Mol. Cell. Biol. 17, 760-769) long-patch mismatch-repair systems. Mismatch repair directed specifically against incorrect bases inserted during semi-conservative DNA replication might efficiently antagonize UV mutagenesis. To test this hypothesis, DNA 51-mers containing site-specific T-T cis-syn-cyclobutane pyrimidine-dimers or T-T pyrimidine-(6-4')pyrimidinone photoproducts, with all four possible bases opposite the respective 3'-thymines in the photoproducts, were analyzed for the ability to compete with radiolabeled (T/G)-mismatched DNA for binding by highly purified human MSH2.MSH6 heterodimer protein (hMutSalpha). Both (cyclobutane-dimer)/AG and ((6-4)photoproduct)/AG mismatches competed about as well as non-photoproduct T/T mismatches. The two respective pairs of photoproduct/(A(T or C)) mismatches also showed higher hMutSalpha affinity than photoproduct/AA "matches"; the apparent affinity of hMutSalpha for the ((6-4)photoproduct)/AA-"matched" substrate was actually less than that for TT/AA homoduplexes. Surprisingly, although hMutSalpha affinities for both non-photoproduct UU/GG double mismatches and for (uracil-cyclobutane-dimer)/AG single mismatches were high, affinity for the (uracil-cyclobutane-dimer)/GG mismatch was quite low. Equilibrium binding of hMutSalpha to DNA containing (photoproduct/base) mismatches and to (T/G)-mismatched DNA was reduced similarly by ATP (in the absence of magnesium).     

Processing of mispaired and unpaired bases in heteroduplex DNA in E. coli

Bacteriophage lambda and phi X 174 DNAs, carrying sequenced mutations, have been used to construct in vitro defined species of heteroduplex DNA. Such heteroduplex DNAs were introduced by transfection, as single copies, into E. coli host cells. The progeny of individual heteroduplex molecules from each infective center was analyzed. The effect of the presence of GATC sequences (phi X 174 system) and of their methylation (lambda system) was tested. The following conclusions can be drawn: some mismatched base pairs trigger the process of mismatch repair, causing a localized strand-to-strand information transfer in heteroduplex DNA: transition mismatches G:T and A:C are efficiently repaired, whereas the six transversion mismatches are not always readily recognized and/or repaired. The recognition of transversion mismatches appears to depend on the neighbouring nucleotide sequence; single unpaired bases (frameshift mutation "mismatches") are recognized and repaired, some equally efficiently on both strands (longer and shorter), some more efficiently on the shorter (-1) strand; large non-homologies (about 800 bases) are not repaired by the Mut H, L, S, U system, but some other process repairs the non-homology with a relatively low efficiency; full methylation of GATC sequences inhibits mismatch repair on the methylated strand: this is the chemical basis of strand discrimination (old/new) in mismatch correction; unmethylated GATC sequences appear to improve mismatch repair of a G:T mismatch in phi X 174 DNA, but there may be some residual mismatch repair in GATC-free phi X 174, at least for some mismatches.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA binding and cleavage selectivity of the Escherichia coli DNA G:T-mismatch endonuclease (vsr protein).

The Escherichia coli vsr endonuclease recognises T:G base-pair mismatches in double-stranded DNA and initiates a repair pathway by hydrolysing the phosphate group 5' to the incorrectly paired T. The gene encoding the vsr endonuclease is next to the gene specifying the E. coli dcm DNA-methyltransferase; an enzyme that adds CH3 groups to the first dC within its target sequence CC[A/T]GG, giving C5MeC[A/T]GG. Deamination of the d5MeC results in CT[A/T]GG in which the first T is mis-paired with dG and it is believed that the endonuclease preferentially recognises T:G mismatches within the dcm recognition site. Here, the preference of the vsr endonuclease for bases surrounding the T:G mismatch has been evaluated. Determination of specificity constant (kst/KD; kst = rate constant for single turnover, KD = equilibrium dissociation constant) confirms vsr's preference for a T:G mismatch within a dcm sequence i.e. CT[A/T]GG (the underlined T being mis-paired with dG) is the best substrate. However, the enzyme is capable of binding and hydrolysing sequences that differ from the dcm target site by a single base-pair (dcm star sites). Individual alteration of any of the four bases surrounding the mismatched T gives a substrate, albeit with reduced binding affinity and slowed turnover rates. The vsr endonuclease has a much lower selectivity for the dcm sequence than type II restriction endonucleases have for their target sites. The results are discussed in the light of the known crystal structure of the vsr protein and its possible physiological role.     

Structural features and hydration of a dodecamer duplex containing two C.A mispairs.

X-ray diffraction techniques have been used to characterise the crystal and molecular structure of the deoxyoligomer d(C-G-C-A-A-A-T-T-C-G-C-G) at 2.5A resolution. The final R factor is 0.19 with the location of 78 solvent molecules. The oligomer crystallises in a B-DNA type conformation with two strands coiled about each other to produce a duplex. This double helix consists of four A.T and six G.C Watson-Crick base pairs and two C.A mispairs. The mismatched base pairs adopt a "wobble" type structure with the cytosine displaced laterally into the major groove, the adenine into the minor groove. We have proposed that the two close contacts observed in the C.A pairing represent two hydrogen bonds one of which results from protonation of adenine. The mispairs are accommodated in the double helix with small adjustments in the conformation of the sugar-phosphate backbone. Details of the backbone conformation, base stacking interactions, thermal parameters and the hydration are now presented and compared with those of the native oligomer d(C-G-C-G-A-A-T-T-C-G-C-G) and with variations of this sequence containing G.T and G.A mispairs.     

Crystal structure and stability of a DNA duplex containing A(anti).G(syn) base-pairs

The synthetic dodecanucleotide d(CGCAAATTGGCG) has been analysed by single-crystal X-ray diffraction techniques and the structure refined to R = 0.16 and 2.25 A resolution, with the location of 94 solvent molecules. The sequence crystallizes as a full turn of a B-DNA helix with ten Watson-Crick base-pairs and two adenine-guanine mispairs. The analysis clearly shows that the mismatches are of the form A(anti).G(syn). Thermal denaturation studies indicate that the stability of the duplex is strongly pH dependent, with a maximum at pH 5.0, suggesting that the base-pair is stabilized by protonation. Three different arrangements have been observed for base-pairs between guanine and adenine and it is likely that A.G mismatch conformation is strongly influenced by dipole-dipole interactions with adjacent base-pairs.     

Chemical reactivity of matched cytosine and thymine bases near mismatched and unmatched bases in a heteroduplex between DNA strands with multiple differences.

The chemical reactivity of matched T and C bases to osmium tetroxide and hydroxylamine near mismatched and unmatched bases in a heteroduplex between two strands of DNA with multiple differences was examined. Data was available for matched bases one or two positions away from 24 mismatches. Reactive bases were found near 16 of the mismatches and were usually one or two bases away. This reactivity is consistent with structural studies indicating perturbation of the duplex around mismatches and will allow another mode of study of the effect of mismatches. The reactivity of these bases was found not to be strongly correlated with mismatch type or GC basepair content of the basepairs around the mismatches. Extra reactivity may have been promoted by the presence of either T or C in the mismatch allowing increased reactivity of nearby T or C. The utility of the phenomenon for the detection of mutations is discussed. Unmatched bases in the heteroduplex also gives rise to reactive matched bases nearby.     

Nearest-neighbor parameters for G.U mismatches: [formula; see text] is destabilizing in the contexts [formula; see text] and [formula; see text] but stabilizing in [formula; see text].

Thermodynamic parameters derived from optical melting studies are reported for duplex formation by a series of oligoribonucleotides containing G.U mismatches. The results are used to determine nearest-neighbor parameters for helix propagation by G.U mismatches. Surprisingly, the [formula; see text] nearest-neighbor free energy increment in unfavorable in the contexts [formula; see text], and [formula; see text] but favorable in the context [formula; see text]. This is a non-nearest-neighbor effect. In contrast, the [formula; see text] free energy increment is favorable and independent of context. Circular dichroism and imino proton NMR spectra of several sequences do not reveal an obvious structural basis for this dichotomy. For example, all the G.U mismatches have two slowly exchanging imino protons. The imino resonances for the G.U mismatches in GGAGUUCC, GUCGUGAC, and CCUGUAGG, however, broaden at lower temperature than the imino resonances for the interior Watson-Crick base pairs. In contrast, the imino resonances for the G.U mismatches in GGAUGUCC remain sharp at high temperature. The improved parameters for G.U mismatches should improve predictions of RNA structure from sequence.     

Influence of pH on the conformation and stability of mismatch base-pairs in DNA

A series of self-complementary dodecanucleotide duplexes containing two symmetrically disposed mismatches have been studied by pH-dependent, ultraviolet light melting techniques. The results indicate that A.C, and C.C mismatches are strongly stabilized by protonation and that the degree of stabilization of the A.C mismatch depends greatly on the flanking bases. In one case, a duplex containing two A.C mismatches is more stable than the native sequence below pH 5.5. The G.A mismatch displays conformational flexibility, with a protonated G(syn).A(anti) base-pair occurring in certain base stacking environments but not in others. The A.A and T.C mismatches are not stabilized at low pH. These solution studies correlate well with predictions based on X-ray crystallographic data.     

Wobble dG X dT pairing in right-handed DNA: solution conformation of the d(C-G-T-G-A-A-T-T-C-G-C-G) duplex deduced from distance geometry analysis of nuclear Overhauser effect spectra

We report below on features of the three-dimensional structure of the d(C-G-T-G-A-A-T-T-C-G-C-G) self-complementary duplex (designated 12-mer GT) containing symmetrical G X T mismatches in the interior of the helix. The majority of the base and sugar protons in the 12-mer GT duplex were assigned by two-dimensional nuclear Overhauser effect (NOESY) spectra in H2O and D2O solution. A set of 92 short (less than 4.5-A) proton-proton distances defined by lower and upper bounds for one symmetrical half of the 12-mer GT duplex were estimated from NOESY data sets recorded as a function of mixing time. These experimental distances combined with nucleotide bond length parameters were embedded into Cartesian space; several trial structures were refined to minimize bond geometry and van der Waals and chirality error. Confidence in this approach is based on the similarity of the refined structures for the solution conformation of the 12-mer GT duplex. The G and T bases pair through two imino-carbonyl hydrogen bonds, and stacking is maintained between the G X T wobble pair and adjacent Watson-Crick G X C pairs. The experimental distance information is restricted to base and sugar protons, and hence structural features such as base pair overlap, glycosidic torsion angles, and sugar pucker are well-defined by this combination of NMR and distance geometry methods. By contrast, we are unable to define the torsion angles about the bonds C3'-O3'-P-O5'-C5'-C4' in the backbone of the nucleic acid.     

Two nicking enzyme systems specific for mismatch-containing DNA in nuclear extracts from human cells.

We have identified two novel enzyme systems in human HeLa nuclear extracts that can nick at specific sites of DNA molecules with base mismatches, in addition to the T/G mismatch-specific nicking enzyme system (Wiebauer, K., and Jiricny, J. (1989) Nature 339, 234-236). One enzyme (called all-type) can nick all eight base mismatches with different efficiencies. The other (A/G-specific) nicks only DNA containing an A/G mismatch. The all-type enzyme can be separated from the T/G-specific and A/G-specific nicking enzymes by Bio-Rex 70 chromatography. Further purification on a DEAE-5PW column separated the A/G-specific nicking enzyme from the T/G-specific nicking enzyme. Therefore, at least three different enzyme systems are able to cleave mismatched DNA in HeLa nuclear extracts. The all-type and A/G-specific enzymes work at different optimal salt concentrations and cleave at different sites within the mismatched DNA. The all-type enzyme can only cleave at the first phosphodiester bond 5' to the mispaired bases. This enzyme shows nick disparity to only one DNA strand and may be involved in genetic recombination. The A/G-specific enzyme simultaneously makes incisions at the first phosphodiester bond both 5' and 3' to the mispaired adenine but not the guanine base. This enzyme may be involved in an A/G mismatch-specific repair similar to the Escherichia coli mutY (or micA)-dependent pathway.     

Structure, dynamics, and thermodynamics of mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2

The structures and hydrogen exchange properties of the mismatched DNA oligonucleotide duplexes d(CCCAGGG)2 and d(CCCTGGG)2 have been studied by high-resolution nuclear magnetic resonance. Both the adenine-adenine and thymine-thymine mismatches are intercalated in the duplexes. The structures of these self-complementary duplexes are symmetric, with the two strands in equivalent positions. The evidence indicates that these mismatches are not stably hydrogen bonded. The mismatched bases in both duplexes are in the anti conformation. The mismatched thymine nucleotide in d(CCCTGGG)2 is intercalated in the duplex with very little distortion of the bases or sugar-phosphate backbone. In contrast, the bases of the adenine-adenine mismatch in d(CCCAGGG)2 must tilt and push apart to reduce the overlap of the amino groups. The thermodynamic data show that the T-T mismatch is less destabilizing than the A-A mismatch when flanked by C-G base pairs in this sequence, in contrast to their approximately equal stabilities when flanked by A-T base pairs in the sequence d(CAAAXAAAG.CTTTYTTTG) where X and Y = A, C, G, and T [Aboul-ela, F., Koh, D., & Tinoco, I., Jr. (1985) Nucleic Acids Res. 13, 4811]. Although the mechanism cannot be determined conclusively from the limited data obtained, exchange of the imino protons with solvent in these destabilized heteroduplexes appears to occur by a cooperative mechanism in which half the helix dissociates.