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The three-dimensional structure of the hairpin formed by d(ATCCTA-GTTA-TAGGAT) has been determined by means of two-dimensional NMR studies, distance geometry and molecular dynamics calculations. The first and the last residues of the tetraloop of this hairpin form a sheared G-A base pair on top of the six Watson-Crick base pairs in the stem. The glycosidic torsion angles of the guanine and adenine residues in the G-A base pair reside in the anti and high- anti domain ( approximately -60 degrees ) respectively. Several dihedral angles in the loop adopt non-standard values to accommodate this base pair. The first and second residue in the loop are stacked in a more or less normal helical fashion; the fourth loop residue also stacks upon the stem, while the third residue is directed away from the loop region. The loop structure can be classified as a so-called type-I loop, in which the bases at the 5'-end of the loop stack in a continuous fashion. In this situation, loop stability is unlikely to depend heavily on the nature of the unpaired bases in the loop. Moreover, the present study indicates that the influence of the polarity of a closing A.T pair is much less significant than that of a closing C.G base pair.

We have previously shown that nuclear DNA of bloodstream from Trypanosoma brucei contains a novel base beta-glucosyl-hydroxymethyluracil, called J. Base J is enriched in minichromosome fractions but not in the minichromosome internal repeats, suggesting the association of J with telomeric DNA. To test whether J is present in the long telomeric (GGGTTA)n repeat arrays, which are 2-26 kb in T.brucei, we have purified these arrays both by hybrid selection and by isolating 2-26 kb fragments from DNA digested with multiple restriction enzymes. We find that in purified telomeric repeats approximately 13% of T is replaced by J, compared to 0.8% in total DNA, and we estimate that approximately 50% of the total J is in these repeats. Highly purified complementary strands of the repeats were obtained by alkaline CsCl equilibrium centrifugation. In the (TAACCC)n strand 14% of T was replaced by J. In the (GGGTTA)n strand approximately 36% of the second T was replaced by J; the first T was not detectably replaced. Modified bases have not been found in telomeric repeats before. How the bulky base J affects telomere function and structure in bloodstream form trypanosomes remains to be determined.

The 3'-5' circular trinucleotide cr(GpGpGp) was studied by means of 1D and 2D high resolution NMR techniques and molecular mechanics calculations. Analysis of the J-couplings, obtained from the 1H and 13C-NMR spectra, allowed the determination of the conformation of the sugar rings and of the 'circular' phosphate backbone. In the course of the investigations it was found that the Karplus-equation most recently parametrized for the CCOP J-coupling constants could not account for the measured J(C4'P) of 11.1 Hz and a new parametrization for both HCOP and CCOP coupling constants is therefore presented. Subsequent analysis of the coupling constants yielded 'fixed' values for the torsion angles beta and delta (with beta = 178 degrees and delta = 139 degrees). The value of the latter angle corresponds to an S-type sugar conformation. The torsion angles gamma and epsilon are involved in a rapid equilibrium in which they are converted between the gauche(+) and trans and between the trans and gauche(-) domain respectively. We show that the occurrence of epsilon in the gauche(-) domain necessitates S-type sugar conformations. Given the aforementioned values for beta, gamma, delta and epsilon the ring closure constraints for the ring, formed by the phosphate backbone can only be fulfilled if alpha and zeta adopt some special values. After energy minimization with the CHARMm force field only two combinations of alpha and zeta result in energetically favourable structures, i.e. the combination alpha (t)/zeta(g-) in case gamma is in a gauche(+) and epsilon is in a trans conformation, and the combination alpha (t)/zeta (g+) for the combination gamma (t)/epsilon (g-). The results are discussed in relation to earlier findings obtained for cd(ApAp) and cr(GpGp), the latter molecule being a regulator of the synthesis of cellulose in Acetobacter xylinum.

Partially methylphosphonate-modified oligodeoxynucleotides were synthesized on solid-phase by employing the easily removable 2-(acetoxymethyl)benzoyl (AMB) group as base-protecting group. Although a rapid AMB deprotection can be accomplished in methanolic potassium carbonate, the lability of the methylphosphonate linkage towards potassium carbonate/methanol excludes the use of this deprotection reagent. Thus, saturated ammonia solution in methanol was investigated as an alternative reagent for AMB removal. It is demonstrated that the combination of the AMB protective group and ammonia/methanol as deprotection reagent significantly improves the synthesis of methylphosphonate-modified DNA fragments. A mild overnight treatment at room temperature is sufficient for complete removal of the AMB group, whereas deprotection of conventionally protected oligonucleotides requires much longer exposure to basic conditions at elevated temperatures.

Two heteronuclear proton-carbon NMR experiments are applied to the DNA-octamer d(TTGGCCAA)2 with carbon in natural abundance. They lead to a complete assignment of the carbon resonances of the sugars and bases. In addition, several heteronuclear coupling constants, proton-carbon as well as proton-phosphorous and phosphorous-carbon, were determined. The information can be obtained in a reasonable measuring time and offers valuable information for a detailed picture of DNA structure.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.

The naturally occurring DNA-nucleopeptide H-Asp-Ser[5'-pAAAGTAAGCC-3']-Glu-OH was prepared via a solid-phase phosphite triester approach using N-2-(tert-butyldiphenylsilyloxymethyl)benzoyl protected nucleosides. The oligonucleotide was linked via the extremely base-labile oxalyl ester anchor to the solid support.

Synthetically prepared ribosylribitol phosphate dimer and trimer analogous to fragments of the capsular polysaccharide of Haemophilus influenzae type b, containing either an amino- or a (masked) thiol-functionalized spacer, were conjugated to protein by two different methods. The thiol-containing carbohydrates were conjugated to tetanus toxoid or H. influenzae outer membrane protein using N-succinimidyl 3-(2-pyridyldithio)propionate. Glutaric dialdehyde was used to conjugate the amino-containing carbohydrates with tetanus toxoid. All conjugates were able to bind antibodies raised against the native bacterial polysaccharide, as determined by competition ELISA. The glutaric dialdehyde conjugate prepared from ribosylribitol phosphate trimer and tetanus toxoid induced a strong polysaccharide-specific antibody response in mice and rabbits.

The naturally occurring RNA-nucleopeptide H-Ala-Tyr[5'-pUUAAAAC-3']-NH2 is prepared via a solid-phase phosphite triester approach using N-SiOMB/O-TBDMS-protected nucleosides. Preliminary 1H-NMR studies show that the peptidyl unit has a remarkable effect on the conformational behaviour of the RNA moiety in the nucleopeptide.

The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.

Before integration of the human immunodeficiency virus (HIV) DNA, two nucleotides are removed from the 3' ends of the viral DNA by the integrase (IN) protein. We studied the chemistry of this reaction, and found that IN mediates site-specific hydrolysis of a phosphodiester bond, resulting in release of a dinucleotide. A class of alcohols (including glycerol, 1,2-propanediol, but not 1,3-propanediol) can also act as nucleophile in this reaction, and likewise the alcoholic amino acids L-serine and L-threonine can be covalently linked to the dinucleotide. No evidence was found for a covalent linkage between the IN protein and this dinucleotide, suggesting that IN directs a single nucleophilic attack of water at the specific phosphodiester bond.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three dimensional solution structure of the non-selfcomplementary oligonucleotide, d(GAGGAGGCACG). d(CGTGCGTCCTC) in which the central base pair is G.G. This is the first structural determination of a G.G mismatch in a oligonucleotide. Two dimensional nuclear magnetic resonance spectra show that the bases of the mismatched pair are stacked into the helix and that the helix adopts a classical B-DNA form. Spectra of the exchangeable protons show that the two guanosines are base paired via their imino protons. For the non-exchangeable protons and for some of the exchangeable protons nuclear Overhauser enhancement build up curves at short mixing times have been measured. These give 84 proton-proton distances which are sensitive to the helix conformation. One of the guanosines adopts a normal anti conformation while the other is syn or close to syn. All non-terminal sugars are C2' endo. These data sets were incorporated into the refinement of the oligonucleotide structure by molecular mechanics calculations. The G.G mismatch shows a symmetrical base pairing structure. Although the mismatch is very bulky many of its features are close to that of normal B-DNA. The mismatch induces a small lateral shift in the helix axis and the sum of the helical twist above and below the mismatch is close to that of B-DNA.

We have previously shown that the pyrimidine oligonucleotide 5'CTTCCTCCTCT (Y11) recognizes the double-helical stem of hairpin 5'GAAGGAGGAGA-T4-TCTCCTCCTTC (h26) by triple-helix formation (1). In this paper, we report the effect on triplex formation of substituting the cytosine residues of Y11 with 5-methylcytosines (5meY11). In addition, we have studied the thermodynamics of the interaction between h26 and 5meY11. The results can be summarised as follows: (i) gel electrophoresis shows that at T = 5 degrees C and pH 5, both Y11 and 5meY11 form DNA triple helices with h26, whereas at pH 6.8 only the methylated strand binds to h26; (ii) pH-stability curves of the DNA triplexes formed from h26 + Y11 and h26 + 5meY11 show that Y11 and 5meY11 are semi-protonated at pH 5.7 and 6.7, respectively. Thus, it is concluded that cytosine methylation expands the pH range compatible with triplex formation by one pH unit; (iii) as the unmethylated triplex (h26:Y11), the methylated one (h26:5meY11) denatures in a biphasic manner, in which the low temperature transition results from the dissociation of 5meY11 from h26. The Tm of the triplex to h26 plus 5meY11 transition is strongly enhanced (about 10 degrees C) by cytosine methylation. A van 't Hoff analysis of denaturation curves is presented; (iv) DSC experiments show that triplex formation between 5meY11 and h26 is characterized by delta H = -237 +/- 25 kJ/mol and delta S = -758 +/- 75 J/Kmol, corresponding to an average delta H of -21 kJ/mol and delta S of -69 J/Kmol per Hoogsteen base pair; (v) the thermodynamic analysis indicates that the extra stability imparted to the triplex by methylcytosine is entropic in origin.

The 5'-(4,4'-dimethoxytrityl) protected 3'-(2-cyanoethoxy)-N,N-diisopropylphosphoramidite of 7-hydro-8-oxo-2'-deoxy-guanosine, the exocyclic amino and lactam functions of which are protected with acetyl and diphenylcarbamoyl groups, respectively, has been prepared from the 8-bromo derivatives of deoxy- and riboguanosine. This synthon, in combination with standard d-nucleoside 3'-(2-cyanoethoxy)-N,N-diisopropylphosphoramidites, was applied successfully to a solid-phase synthesis. Well-defined oligodeoxyribonucleotides containing a 7-hydro-8-oxo-2'-deoxyguanosine residue at predetermined positions were obtained after deprotection with methanolic ammonia and purification by gel filtration.

Hairpin structures formed by seven DNA inverted repeats have been studied by PAGE, UV(CD)-spectroscopy and nuclease cleavage. The hairpins consisted of (CG)3 stems and loops of 2, 3 and 4 residues. Thermal stabilities (Tm) have been determined in low and high ionic strength buffers, where the hairpins were structured in the B- and Z-DNA form respectively. The thermodynamic parameters of hairpin formation have been obtained by a two-state analysis of the hairpin-coil transitions. It is found that, on increasing the number of bases in the loop from 2 to 3 and 4, the Tms of the B-hairpins decrease, whereas the Tms of the same hairpins in the Z-form increase. This confirms previous evidence (1,2) that in a hairpin molecule the size and structure of the loop are modulated by the conformation of the helical stem. Moreover, B-hairpins with loops comprising 2, 3 and 4 bases have been digested with the single-strand-specific nuclease from mung bean. In our experimental conditions (0 degrees C) the nuclease preferentially cleaves the unbonded nucleotides of the loops. However, the rates of loop hydrolysis, which roughly follow a first-order kinetics, markedly depend on the size of the loop. At a ratio of 3 enzyme units/micrograms DNA, the half-lives of hairpins which are expected to form loops of 4, 3 and 2 residues are 90, 145 and 440 minutes respectively. Thermostability and enzymatic digestion data suggest that two-membered loops can be formed in B-hairpins but not in Z-hairpins.

The preparation of the nucleopeptide H-Phe-Tyr-(pATAT)-NH2 could be realized via a solid phase phosphitetriester approach and by using the protected protecting group 2-(tert-butyldiphenylsilyoxymethyl)-benzoyl for the masking of the N6-amino function of deoxyadenosine. The latter protecting group can be removed under mild conditions with fluoride ion.

The interactions between a novel antitumor drug nogalamycin with the self-complementary DNA hexamer d(CGTACG) have been studied by 500 MHz two dimensional proton nuclear magnetic resonance spectroscopy. When two nogalamycins are mixed with the DNA hexamer duplex in a 2:1 ratio, a symmetrical complex is formed. All non-exchangeable proton resonances (except H5' & H5") of this complex have been assigned using 2D-COSY and 2D-NOESY methods at pH 7.0. The observed NOE cross peaks are fully consistent with the 1.3 A resolution x-ray crystal structure (Liaw et al., Biochemistry 28, 9913-9918, 1989) in which the elongated aglycone chromophore is intercalated between the CpG steps at both ends of the helix. The aglycone chromophore spans across the GC Watson-Crick base pairs with its nogalose lying in the minor groove and the aminoglucose lying in the major groove of the distorted B-DNA double helix. The binding conformation suggests that specific hydrogen bonds exist in the complex between the drug and guanine-cytosine bases in both grooves of the helix. When only one drug per DNA duplex is present in solution, there are three molecular species (free DNA, 1:1 complex and 2:1 complex) in slow exchange on the NMR time scale. This equilibrium is temperature dependent. At high temperature the free DNA hexamer duplex and the 1:1 complex are completely destabilized such that at 65 degrees C only free single-stranded DNA and the 2:1 complex co-exist. At 35 degrees C the equilibrium between free DNA and the 1:1 complex is relatively fast, while that between the 1:1 complex and the 2:1 complex is slow. This may be rationalized by the fact that the binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. A separate study of the 2:1 complex at low pH showed that the terminal GC base pair is destabilized.

DNA oligonucleotides with the sequence corresponding to the plus strand origin of replication of the filamentous bacteriophage M13 are studied. Biochemical structure probing and UV melting studies, supplemented with initial NMR experiments, are used to investigate structural features of a 51-nucleotides long synthetic oligonucleotide and two oligonucleotides that are integral parts of this latter molecule. The results demonstrate the feasibility and complementarity of the use of methidiumpropyl.EDTA-Fe(II) and nuclease S1 in the structural analysis of small oligonucleotides. The bacteriophage origin region appears to comprise two hairpins. The first hairpin, which contains a cleavage site for the bacteriophage gene II protein, has a large and probably flexible loop. NMR as well as UV melting studies demonstrate that the second hairpin contains a stable three-membered loop. Both hairpins are present in the 51-mer, which forms a stable tertiary structure.

We developed a simian virus 40 based shuttle vector system to study the molecular consequences of distinct carcinogen-induced DNA lesions in human cells. To establish the mutagenicity of O4-ethylthymine adducts, oligonucleotides carrying a single O4-ethylthymine adduct at a unique position were ligated into the vector molecules. Following replication in HeLa cells on average 23% of the progeny molecules carried a mutation in the region of modification. The vast majority of these mutations represented single T----C transitions at the position of the modified base, most probably as a consequence of mispairing of the O4-ethylthymine residues during replication. To a minor extent the O4-ethylthymine adduct may also induce T----A transversions or double point mutations. The in vivo mutation frequency of the adduct was found to be comparable to that of a C-A mismatch at the same position, but was lower than that expected from in vitro experiments with adducted DNA templates and purified DNA polymerases.

Evidence will be presented to show that the reagents 3a-c (X = O) obtained by the reaction of 2-chlorophenylphosphorodichloridate with 1-hydroxybenzotriazole or 1-hydroxy-6-trifluoromethyl (6-nitro) benzotriazoles, respectively, do not give, under normal coupling conditions, phosphorylation of the lactam functions in uracil or guanine. Reagent 3b (X = O) proved to be very convenient for the in situ formation of 3'-5'-phosphotriester linkage. The latter will be illustrated by the synthesis of several RNA fragments.

The hexamer d(m5CGTAm5CG) crystallizes as a left-handed Z-DNA molecule in a isomorphous lattice to d(m5CGm5CGm5CG). The three-dimensional structure has been determined by x-ray diffraction and refined to a final R value of 16% at 1.2A resolution. The molecular structure is similar to the Z-DNA structure seen in d(m5CGm5CGm5CG) and d(CGCGCG). The presence of TpA sequence in the middle of the molecule has resulted in slight changes in the helical twist angles, base pair stacking and twist angles of the base pair planes. The hydration structure is significantly changed from both of methylated and non-methylated d(CGCGCG). A large hole which has no water molecule tightly bound to the DNA molecule is found inside of the deep groove of the DNA helix at the TpA sequence. This deterioration of the hydration structure is associated with destabilization of the Z-DNA structure. These characteristic features of the structure are compared with another Z-DNA hexamer contains TpA sequence, d(br5CGTAbr5CG).

Phosphorylation of the easily accessible 3',5'-diesters 1a-d with diphenyl phosphorochloridate, followed by selective 5'-deacylation, affords the phosphotriester derivatives 2a-d in good yields. Alkaline treatment of 2a-d results in the formation of the 2',3'-cyclic phosphates (3a-d). The usefulness of the phosphotriester derivatives 2a-d is also demonstrated in the synthesis of the nucleotidyl-(3'-5')nucleoside 3'-phosphates U-Up (10a), U-Ap (11a), U-Cp (12a) and A-Gp (13a). The fully protected dinucleoside diphosphates 5c-8c, prepared by the phosphotriester method, are deprotected in two ways: (a) by a purely chemical method, affording the dinucleoside diphosphates in a circa one to one mixture of 2'- and 3'- isomers, 10b-13b and 10a-13a, respectively, and (b) by a mixed chemical-enzymatical approach which gives the pure 3'-phosphates (10a-13a).

The hydrogen-bonded imino protons of a 14 base pair double-stranded DNA fragment comprising one half of the lac operator of E. coli were investigated by 360 MHz H NMR. From combined melting studies of this synthetic 14 b.p. fragment and its two constituent 7 b.p. fragments a nearly complete assignment for the low-field proton resonances was obtained. The experimental spectra are compared with calculated spectra and with the spectrum of a 51 b.p. DNA restriction fragment from E. coli containing the complete lac operator. Structural information on these oligonucleotides is presented. This study is a prerequisite for future 1H NMR investigations of the interaction of the lac operator with the lac repressor.

A new and attractive phosphorylation procedure which allows the introduction, via phosphotriester intermediates, of 5'-phosphate functions of DNA fragments is described. The method is based on the activation of bifunctional phosphorylating agents with 1-hydroxybenzotriazole. The approach will be exemplified by the synthesis of pACGC using four different 5'-phosphotriester intermediates.

NMR studies were carried out on various equimolar mixtures consisting of a combination of oligomers: d(ACGGCT) (I). d(pACGGCT) (Ia), d(TGCAGT) (II), d(AGCCGTACTGCA) (III), d(TGCAGTACGGCT) (IV). It is shown that I + II + III (MI) and Ia + II + III (M2) form stable duplexes with nicks in the centre of the respective double helices. A close analysis of the NOESY experiments of M1 and M2 revealed that these fragments form B-DNA type duplex structures. A comparison of the chemical-shift data of the nicked duplexes with those of the intact duplex of III + IV (M3) demonstrated that only small local distortions occur when a nick is introduced. The chemical-shift profiles of M1 and M3 were used to obtain the thermodynamic data for the duplex/coil transitions. The profiles of M1 were analysed by means of a new thermodynamic model (TRIDUP). From the calculated thermodynamic data of M1 and M3 it is concluded that the melting behaviour of M1 occurs cooperatively. A ligation experiment demonstrated that the relatively small substrate (M2) was almost completely joined after an overnight incubation at 14 degrees C.

The synthesis of two fully-protected hexanucleotides (11a and 11b) via a phosphotriester approach, which is based on the use of two types of protecting groups for the internucleotide linkages, i.e. one 2,2,2-tribromo-ethyl at the 5'-terminus and four 2-chlorophenyl groups for the remaining linkages, is reported. The hexanucleotides 11a and 11b, assembled via a block-wise two-step phosphotriester method, can be deblocked conveniently to give the two hexamers 12a and 12b containing only 3'leads to5' internucleotide linkages.

Carbon-13 and proton NMR spectra of a series of oligodeoxynucleotides (d(CT), d(CC), d(TA), d(AT), d(CG), d(GC), d(AG), d(AAA), d(TATA) and d(GGTAAT] were measured at various temperatures. The three coupling constants that are related to the magnitude of backbone angle epsilon (J(C4'-P), J(C2'-P) and J(H3'-P] are analyzed in terms of a three-state equilibrium about this bond. Two epsilon (trans) angles occur, which differ in magnitude depending on the conformation (N or S) of the adjoining deoxyribose ring. The S-type deoxyribose ring is associated with a smaller epsilon (trans) angle: epsilon (t,S) = 192 degrees. The N-type deoxyribose ring is associated with a larger epsilon (trans) angle epsilon (t,N) = 212 degrees. The third rotamer participating in the conformational equilibrium, is a gauche(-) (epsilon (-] conformer and occurs exclusively in combination with the S-type sugar ring (epsilon (-,S) = 266 degrees). Within the limits of experimental error, the magnitude of these three angles appears to be independent of the particular base sequence, except in the case of d(CG) where a slightly larger epsilon (t,S) angle (197 degrees) is indicated. A simple equation is proposed which may be used to calculate the population of epsilon (t,S) conformer in cases where only J(H3'-P) is known.

The complete and unequivocal assignment of the 24 ribose proton signals of m6(2)A(1)-U(2)-m6(2)(3)-U(4) by means of 500 MHz NMR spectroscopy at 17 degrees C is given. this assignment is based on scrupulous decoupling experiments carries out at various temperatures. Analysis of the observed chemical shifts and coupling constants of the tetramer shows that the two fragments -m6(2)A(3)-U(4) comprising the 3'-end occur mainly in the classical right-handed stack conformation, whereas the 5'-end the -U(2)- residue appears bulged out in favour of a less well-defined stacking interaction between the bases m6(2)A(1)-and -m6(2)A(3)-. Conformational populations about each of the torsional degrees of freedom along the backbone are discussed. A modernized version of pseudorotation analysis is used to delineate the conformational behaviour of the four ribose rings.

A comparative nuclear magnetic resonance study of the hydrogen-bonded imino protons in a series of synthetic DNA fragments is presented. The fragments ATCCTA(Tn)TAGGAT are in principle capable of forming either a self-complementary hairpin loop structure (monomer form) or an interior loop structure (dimeric form). It has been shown, that for n = 1 only the dimer structure is present in aqueous solution, whereas the exclusive existence of the hairpin loop structure is indicated for n = 3, 4 & 5. Surprisingly, for n = 2 two different structures appear to be present in solution. Concentration studies show that both monomers and dimers exist side by side in this case. Hairpins as well as interior loops form extra "melting sites" in addition to the wellknown fraying phenomenon at the terminus of the double helix.

The synthetic DNA fragment (formula, see text) (corresponding to nucleotides 4299-4314 of the phi X DNA sequence) was cloned into either the AmpR gene or the KmR gene of plasmid pACYC 177. The DNA sequence of the KmR gene around the insertion site was determined by nucleotide sequence analysis of the pACYC 177 FnudII restriction DNA fragment N6 (345 b.p.). Of five selected plasmid DNAs, which contained inserted DNA sequences in the antibiotic resistance genes, the nucleotide sequences at and around these insertions were determined. Two recombinant plasmids (pFH 704 and pFH 614) contain the hexadecamer sequence in tandem (tail-to-tail and tail-to-head). In the recombinant plasmids pFH 812, pFH 903 and pFH 807 the DNA sequence homology with the phi X origin region was 14 (No. 4300-4313), 16 (No. 4299-4314) and 20 nucleotides (No. 4299-4318), respectively. None of the supercoiled recombinant plasmid DNAs is nicked upon incubation with phi X gene A protein. Moreover, the recombinant plasmid RFI DNAs cannot act as substitutes for phi X RFI DNA in the in vitro (+) strand synthesizing system. It has been shown earlier that single-stranded DNA, which contains the decamer sequence CAACTTGATA is efficiently nicked by the phi X gene A protein. The present results indicate that for nicking of double-stranded supercoiled DNA nucleotide sequence homology with the phi X origin region of more than 20 nucleotides is required. These results suggest a model for initiation of phi X RF DNA replication, which involves the presence of the recognition sequence CAACTTGATA of the phi X gene A protein as well as a second specific nucleotide sequence which is required for the binding of the phi X gene A protein. This binding causes local unwinding of the DNA double helix and exposure of the recognition sequence in a single-stranded form, which then can be nicked by phi X gene A protein.

A 9.2 kb segment of the maxi-circle of Trypanosoma brucei mitochondrial DNA contains the genes for cytochrome c oxidase subunits I and II (coxI and coxII) and seven Unassigned Reading Frames ("URFs"). The genes for coxI and coxII display considerable homology at the aminoacid level (38 and 25%, respectively) to the corresponding genes in fungal and mammalian mtDNA, the only striking point of divergence being an unusually high cysteine content (about 4.5%). The reading frame coding for cytochrome c oxidase subunit II is discontinuous: the C-terminal portion of about 40 aminoacids, is present in the DNA-sequence in a -1 reading frame with respect to the N-terminal moiety. URF5, 8 and 10, show a low but distinct homology (about 20%) to mammalian mitochondrial URF-1, 4 and 5, respectively. In URF5, the first AUG is found at codon 145, whereas extensive homology to mammalian URF-1 sequences occurs upstream of this position. The possibility exists that UUG can serve as an initiator codon. URF7 and URF9 have a highly unusual aminoacid composition and do not possess AUG or UUG initiator codons. These URFs probably do not have a protein-coding function. The segment does not contain conventional tRNA genes.

The 30 ribose proton resonances of the pentaribonucleoside tetraphosphate m6(2)AUm6(2)AUm6(2)A have been assigned unequivocally by means of spin-echo-correlated spectroscopy, 2D J-resolved spectroscopy and Nuclear Overhauser difference spectroscopy, carried out at 500 MHz. A detailed comparison of the conformational properties of the title compound with its constituent fragments m6(2)AUm6(2)AU, m6(2)AUm6(2)A, m6(2)AU and the relevant monomers is given. Chemical shift data indicate the existence of a doubly "bulged out" conformer, in which the two interior U-fragments are not involved in regular nearest neighbour stacking interactions. The coupling constants of the ribose-ring are interpreted in terms of the N/S equilibrium, and population distributions along the backbone angles beta and gamma are presented. The combined data suggest a strong similarity between the 5'-terminal triplets in m6(2)AUm6(2)AUm6(2)A, m6(2)AUm6(2)AU and m6(2)AUm6(2)A2.

A study on the conformation of the title compound, C-C-A, and on its constituent dinucleotides is presented. 1H-NMR spectra at 360 and 500 MHz were completely assigned by decoupling experiments. Computer simulation of the spectra yielded precise proton-proton and proton-phosphorus coupling constant values. The coupling constants are analyzed in terms of torsion angles and of N- and S-type sugar pucker. 31P-NMR spectra gave some information about P-O backbone torsion angles alpha and zeta. CD spectroscopy was used to obtain insight in the base-base interaction. The C(1) and C(2) unit in C-C-A show normal preference for N-type conformation of the sugar ring, whereas the A(3) residue appears rather biased towards the S-conformation. The zeta and alpha backbone torsion angles in the C-C phosphodiester linkage in C-C-A appear to assume normal g-, g- conformation, the zeta, alpha combination in the C-A linkage is proposed to have a g+, t conformation. In the C-C fragment in C-C-A a regular stack is indicated; it is suggested that the C-A part adopts an unusual antiparallel base stack.

The bifunctional and crystalline phosphorylating agent morpholino-0,0-bis[1-benzotriazolyl]phosphate has been used for the preparation of a 3',5'-bis-phosphotriester intermediate of thymidine. The latter has been converted into ppTppp by the following consecutive steps; removal of the benzotriazolyl group followed by the addition of phosphoric acid and removal of the 2-(4-nitrophenyl)-ethyl group followed by the addition of pyrophosphoric acid.

The synthetic oligodeoxyribonucleotide pCGAAAGACTACAC has been applied as a site-specific mutagen to introduce a T leads to G transversion mutation at nucleotide position 1223 of the M13 DNA sequence. The in vitro-induced conversion of a TAT codon into a TAG at this position resulted in gene IX mutants with an amber mutant character thereby confirming that this reading frame defines a gene of an essential phage protein. The gene IX amber mutants obtained grew well on SuI (Ser) and SuIII (Tyr) suppressing strains but could not be propagated on SuII (Gln) and SuVI (Leu) strains. Complementation studies show that amber mutants in genes V and VII exert a polar effect on gene IX expression suggesting that these three contiguous genes form an operon. In addition, we demonstrate the in vitro synthesis of gene IX-protein in a coupled transcription-translation system.

Analytical and experimental evidence will be presented to show that addition of a slight excess of 2-chlorophenyl-0,0-bis[1-benzotriazolyl]phosphate to a properly 2',5'-protected ribonucleoside gives an intermediate 3'-phosphotriester, which, after addition of a 2'-protected ribonucleoside, affords a dimer containing solely a 3'-5'-internucleotide linkage. The above phosphorylation procedure has been used in the synthesis of the pentamer UpApCpGpC.

Evidence will be presented to show that the allyl group is suitable for the protection of a 3'-terminal phosphodiester function. The latter will be demonstrated by the synthesis, via a phosphotriester approach, of two cyclic tetraribonucleotides [r(AAAA) and r(UAMe2UAMe2)], two cyclic hexadeoxyribonucleotides [d(CGCGCG) and d(TAAAAA)] and a cyclic octadeoxyribonucleotide [d(CGTGCGTG)].

The deoxyribose hexanucleoside pentaphosphate (m5dC-dG)3 has been studied by 500 MHz 1H NMR in D2O (0.1 M NaCl) and in D2O/deuterated methanol mixtures. Two conformations, in slow equilibrium on the NMR time scale, were detected in methanolic solution. Two-dimensional nuclear Overhauser effect (NOE) experiments were used to assign the base and many of the sugar resonances as well as to determine structural features for both conformations. The results were consistent with the an equilibrium in solution between B-DNA and Z-DNA. The majority of the molecules have a B-DNA structure in low-salt D2O and a Z-DNA structure at high methanol concentrations. A cross-strand NOE between methyl groups on adjacent cytosines is observed for Z-DNA but not B-DNA. The B-DNA conformation predominates at low methanol concentrations and is stabilized by increasing temperature, while the Z-DNA conformation predominates at high methanol concentrations and low temperatures. 31P NMR spectra gave results consistent with those obtained by 1H NMR. Comparison of the 31P spectra with those obtained on poly(dG-m5dC) allow assignment of the lower field resonances to GpC in the Z conformation.

We have recorded NOESY spectra of two non-selfcomplementary undecanucleotide duplexes. From the observed NOEs we do not detect any significant distortion of the helix when a G-C pair is replaced by a G-T pair and the normal interresidue connectivities can be followed through the mismatch site. We conclude that the 2D spectra of the non-exchangeable protons do not allow differentiation between a wobble or rare tautomer form for the mismatch. NOE measurements in H2O, however, clearly show that the mismatch adopts a wobble structure and give information on the hydration in the minor groove for the G-T base pair which is embedded between two A-T base pairs in the sequence.

Synthetic oligonucleotides, DNA ligase and DNA polymerase were used to construct double-stranded DNA fragments homologous to the first 25, 27 or 30 b.p. of the origin of replication of bacteriophage phi X174 (nucleotides 4299-4328 of the phi X174 DNA sequence). The double-stranded DNA fragments were cloned into the unique SmaI or HindIII restriction sites in the kanamycin-resistance gene of pACYC177 (AmpR, KmR). Recombinant plasmids were picked up by colony hybridization. DNA sequencing showed that not only recombinant plasmids with the expected insert were formed, but also recombinant plasmids with a shorter insert. Recombinant plasmids with an insert homologous to the first 24, 25, 26, 27, 28 or all 30 b.p. of the phi X174 origin region were thus obtained. Supercoiled plasmids containing a sequence homologous to the first 27, 28 or 30 b.p. of the phi X174 origin region are nicked by the phi X174 gene A protein. However, the other supercoiled plasmids are not nicked by the phi X174 gene A protein. These results show that the first 27 b.p. of the phi X174 origin region are sufficient as well as required for the initiation step in phi X174 RF DNA replication, i.e. the cleavage by gene A protein.

Use of the protecting groups di-n-butylaminomethylene,2-nitrophenylsulfenyl and the ester of 2-(hydroxymethyl)-9,10-anthraquinone, enabled us for the first time to prepare nucleopeptide fragments containing 2'-deoxyguanosine and a free carboxylic acid group.

The monofunctional phosphitylating reagents bis-(N,N-diethylamino)chlorophosphine and salicylchlorophosphine have been applied for the preparation of H-phosphonates of the amino acids serine, threonine and tyrosine. Experimental evidence showed that the latter reagent was less effective for the synthesis of a tyrosine H-phosphonate. The amino acids (peptide) H-phosphonates of serine or threonine proved to be suitable starting compounds for the formation of a phosphate diester bond with the 5'-OH of a d-nucleoside derivative using pivaloyl chloride as the activating reagent.

Phosphorylation of N-acyl-5'-O-DMTr-d-nucleosides, or similarly protected DNA-dimers having a free 3'-OH group, with 2-chlorophenyl-0,0-bis(1-benzotriazolyl)phosphate affords reactive 3'-phosphotriester derivatives. The latter intermediates can be used, without further purification, for the synthesis of DNA-fragments on the controlled pore glass/long chain alkylamine support. Further, 2-cyano-1,1-dimethylethoxy dichlorophosphine proved to be very suitable for the preparation of 5'-phosphorylated DNA-fragments on the same type of solid support.

In 4M NaCl solutions (dC-dG)n (n = 3,4; approximately 9 mM) exist as a mixture o +/- B and Z forms. The low and high field components of two 31P NMR resonances originating from internal phosphodiester groups are assigned to the GpC and CpG linkages, respectively. Low temperatures stabilize the Z-forms, which completely disappear above 50 degrees C (n = 3) and 65 degrees C (n = 4). delta H = -44 and -17 kJ/mol for B to Z transition in the hexamer and octamer duplexes, respectively. Temperature dependent changes (0-50 degrees C range) in the spin-lattice relaxation times at 145.7 MHz are distinctly different for the 31P nuclei o +/- GpC and CpG groups. The relaxation data can be explained by assuming that the GpC phosphodiester groups undergo more local internal motion than do the CpG groups.

Two members of the quinoxaline antibiotic family, echinomycin and triostin A, form crystals complexed to a DNA fragment with the sequence d(CpGpTpApCpG). The crystal structure of both complexes was solved by X-ray diffraction to near-atomic resolution. The two structures are similar to each other with differences in some details due to the shorter cross bridge of echinomycin. Both molecules act as bis intercalators surrounding the d(CpG) sequence at either end of the double helix. Alanine forms sequence-specific hydrogen bonds to guanines in the minor groove. The two central AT base pairs are held together by Hoogsteen base pairing with adenine in the syn conformation in both complexes. An octahedrally hydrated magnesium ion is found in the crystal lattice that plays an important role in organizing the lattice as well as stabilizing the complex by hydrogen bonding both to base pairs of DNA and to the quinoxaline ring nitrogen atoms in the major groove side of the DNA double helix. A functional description of the various amino acids in quinoxaline antibiotics is given, together with possible modifications that might affect biological activity.

We have used in vitro site-directed mutagenesis with synthetic DNA oligonucleotides to introduce single nucleotide mutations in yeast mtDNA. In addition to the expected DNA alterations we also recovered with high frequency mutants with large deletions and insertions which arose through interaction with the synthetic DNA fragment. Characterization of a number of these by DNA sequence analysis has permitted reconstruction of the mutagenic events. In all cases, the DNA fragment had base paired with non-adjacent DNA sequences sometimes more than 1000 nucleotides apart from each other on the target strand. The products of such interactions cannot be avoided due to the non-stringent annealing conditions during complementary DNA strand synthesis. However, deliberate mispairing can be directed precisely, as shown by our ability to specifically delete the 1143-bp intron from the yeast mitochondrial gene coding for large ribosomal RNA with a synthetic DNA fragment consisting of the sequence of the exon borders flanking the intron.

NMR and model-building studies were carried out on the duplex d(CTGGTGCGG).d(CCGCCCAG), referred to as (9+8)-mer, which contains an unpaired thymidine residue. Resonances of the base and of several sugar protons of the (9+8)-mer were assigned by means of a NOESY experiment. Interresidue NOEs between dG(4) and dT(5) as well as between dT(5) and dG(6) provided evidence that the extra dT is stacked into the duplex. Thermodynamic analysis of the chemical shift vs temperature profiles yielded an average TmD value of 334 K and delta HD of -289 kJmol-1 for the duplex in equilibrium random-coil transition. The shapes of the shift profiles as well as the thermodynamic parameters obtained for the extra dT residue and its neighbours again indicate that the unpaired dT base is incorporated inside an otherwise intact duplex. This conclusion is further supported by (a) the observation of an imino-proton resonance of the unpaired dT; (b) the relatively small dispersion in 31P chemical shifts (approximately 0.5 ppm) for the (9+8)-mer, which indicates the absence of t/g or g/t combinations for the phosphate diester torsion angles alpha/zeta. An energy-minimized model of the (9+8)-mer, which fits the present collection of experimental data, is presented.

The activation of ras genes in naturally occurring tumors has, thus far, been found to be due to mutations in codon 12 or 61 resulting in single amino acid substitutions. We have used highly labeled synthetic oligonucleotides to detect mutations in these codons and to determine the exact position of the mutation. Using this approach we have found three different mutations in codon 61 of the N-ras gene of various human tumor cell lines. In the fibrosarcoma line HT1080 the first nucleotide of the codon is mutated; in the promyelocytic line HL60 the second and in the rhabdomyosarcoma line RD301 the third nucleotide. For RD301 this implies that the normal glutamine residue at position 61 is replaced by histidine. In addition to the mutated N-ras gene the three cell lines have a normal N-ras gene which is indicative of the dominant character of the mutations.

Dinucleoside methylphosphonates can easily be prepared starting from properly protected d-nucleosides and the bifunctional phosphorylating reagent methyl-O,O-bis(1-benzotriazolyl)phosphate. Separation of the diastereoisomers of 5'-DMTR-d-Ap(Me)T-3'-lev affords optically pure dinucleoside methylphosphonates which, after removal of the 3'-levulinoyl group, have been used for the synthesis of the two optically pure diastereoisomers of the hexamer d-CpGpAp(Me)TpCpG. Further, a one-pot procedure for the preparation of uridine-3',5'-cyclic methylphosphonate will be described. We also found that 3',5'-methylphosphonate linkages in RNA are not stable towards mild acid treatment.