The crystal structure of the oligonucleotide d(CGCAAATTO8GGCG), containing the chemically modified base 8-hydroxydeoxyguanine (O8G), has been determined at 2.5-A resolution and refined to a crystallographic R-factor of 16.8%. The B-type DNA helix contains standard Watson-Crick base pairs except at the mismatch sites, where O8G adopts a syn conformation and forms hydrogen bonds to adenine in the anti conformation. The thermodynamic stability of the duplex was found by UV melting techniques to be intermediate between the native oligonucleotide d(CGCAAATTTGCG) and an oligonucleotide containing A.G mispairs d(CGCAAATTGGCG). Comparison of the structure of the O8G(syn).A(anti) base pair with those of Watson-Crick base pairs has given a reason why O8G.A base pairs are not well repaired by DNA proofreading enzymes.

The hairpin ribozyme is a self-cleaving motif found in the negatives strand of the satellite RNA of some plant viruses. In its natural context, the ribozyme comprises four helices, two of which contain conserved formally unpaired loops, that are adjacent arms of a four-way RNA junction. We show that the arms that would carry these loops are brought close together in the global conformation of the isolated junction. Using fluorescence resonance energy transfer, we demonstrate a two-magnesium ion-dependent conformational transition of the complete ribozyme that brings the loopbearing arms into close physical proximity. The ribozyme is active as a four-way junction, and the rate of cleavage may be modulated by the conformation of the four-way junction.

The self-complementary dodecanucleotide d [CGC(m6OAATTTGCG]2 (where m6G is 0'-methylguanine), which contains two m6G-T base pairs, has been analyzed by x-ray diffraction methods and the structure has been refined to a residual error ofR = 0.185 at 2.0-A resolution. The m6G-T mispair closely resembles a Watson-Crick base pair and there are very few structural differences between the m6G-T duplex and the native analogue. The similarity between the m6G-T base pair and a normal G-C base pair explains the failure of mismatch repair enzymes to recognize and remove this mutagenic lesion. A series of ultraviolet melting studies over a wide pH range on a related dodecamer indicate that the m6G-C mispair can exist in two conformations; one is a wobble pair and the other is a protonated Watson-Crick pair. The former, which predominates at physiological pH, will be removed by normal proofreading and repair enzymes, whereas the latter is likely to escape detection. Hence, the occasional occurrence of the protonated m6G-C base pair may explain why the presence of m6G in genomic DNA does not always give rise to a mutation.The initial stages of chemical carcinogenesis frequently involve the interaction ofgenotoxic agents with DNA to produce covalent modifications in the form of DNA adducts (1-3). An important example of this is the alkylation of the o6 position of guanine residues in DNA resulting from exposure to methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (4) and methyl methanesulfonate and N-methyl-N-nitrosourea (5). The presence of 06-methylguanine (m6G) constitutes a mutagenic lesion that is known to specifically induce G-C to A-T transition mutations (6) and it has been established that protooncogenes can be converted to oncogenes by such a process (7). Hence, the formation of the m6G-T base pair during replication can give rise to a carcinogenic lesion (8,9). In recent years, the biochemical processes involved in chemically induced carcinogenesis have been studied in considerable depth. However, to understand further the mechanisms of mutagenesis, it is necessary to analyze precisely the molecular details of the lesions produced when genotoxic agents interact with DNA. With this overall objective in mind, we have determined the structure of such a lesion, the m6G-T base pair in a B-DNA duplex. § MATERIALS AND METHODSAll oligonucleotides were synthesized by the solid-phase method on an ABI model 380B DNA synthesizer using cyanoethyl phosphoramidite monomers. For those containing m6G, the following protocol was observed: The 5'-dimethoxytrityl-N2-isobutyryl-06-methyldeoxyguanosine 3'-cyanoethyl phosphoramidite monomer was utilized to introduce 06-methyldeoxyguanosine and the fully assembled oligonucleotide was cleaved from the solid support and deprotected in a 5% solution of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in anhydrous methanol for 2 weeks at ambient temperature in an atmosphere of nitrogen (10). At no time was the oligonucleotide exposed to ammonia, as this can lead to the slow conver...

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Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2'-hydroxyl with a 2'-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2'-substituent and 5'-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid-base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3'-5') 5'-thio-5'-deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 x 10(-)(6) s(-)(1) at pH 6 to 3200 x 10(-)(6) s(-)(1) at pH 9, indicating that attack on the phosphorus by the 2'-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3'-5') 5'-amino-5'-deoxyuridine (UpnU) hydrolysis fell from a value of 1802 x 10(-)(6) s(-)(1) at pH 5 to 140 x 10(-)(6) s(-)(1) at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2'-amino-2'-deoxyuridylyl (3'-5') 5'-thio-5'-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 x 10(-)(6) s(-)(1) and 138 x 10(-)(6) s(-)(1). The hydrolysis of 2'-amino-2'-deoxyuridylyl (3'-5') 5'-amino-5'-deoxythymidine (amUpnT) rose from 17 x 10(-)(6) s(-)(1) at pH 11.5 to 11 685 x 10(-)(6) s(-)(1) at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5'-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg(2+), Zn(2+), and Cd(2+) ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis-Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k(cat).

Oxygen intercalation into a pyrochlore at room temperature is reported. A simple chemical route was employed. Previously only perovskite or a few closely related phase have demonstrated an ability to act as hosts for such intercalation. The specific system studied was the interstitial solid solution Ce 2 Zr 2 O 7+x (0 e x e 0.36). Neutron diffraction reveals that interstitial oxygen enters the tetrahedral 8b sites (space group Fd3 hm), which are empty in stoichiometric pyrochlore, but displacement of existing oxygen from the tetrahedral 8a sites also occurs. The lattice contracts on intercalation due to oxidation of Ce 3+ . The changes in structure and the diffusion pathways for oxygen are discussed.

The natural form of the hairpin ribozyme consists of a four-way RNA junction of which the single-stranded loop-carrying helices are adjacent arms. The junction can be regarded as providing a framework for constructing the active ribozyme, and the rate of cleavage can be modulated by changing the conformation of the junction. We find that the junction-based form of the hairpin ribozyme is active in magnesium, calcium, or strontium ions, but not in manganese, cadmium, or sodium ions. Using fluorescence resonance energy transfer experiments, we have investigated the global structure of the ribozyme. The basic folding of the construct is based on pairwise helical stacking, so that the two loop-carrying arms are located on opposite stacked helical pairs. In the presence of magnesium, calcium, or strontium ions, the junction of the ribozyme undergoes a rotation into a distorted antiparallel geometry, creating close physical contact between the two loops. Manganese ions induce the same global folding, but no catalytic activity; this change in global conformation is therefore necessary but not sufficient for catalytic activity. Fitting the dependence of the conformation on ionic concentration to a two-state model suggests that cooperative binding of two ions is required to bring about the folding. However, further ion binding is required for cleavage activity. Cobalt hexammine ions also bring about global folding, while spermidine generates a more symmetrical form of the antiparallel structure. Cadmium ions generate a different folded form, interpreted in terms of close loop-loop association while the junction is unfolded. Sodium ions were unable to induce any folding of the ribozyme, which remained slightly parallel. These results are consistent with a folding process induced by the binding of two group IIA metal ions, distributed between the junction and the loop interface.

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