High-resolution structure of a mutagenic lesion in DNA.

Abstract

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...

The structural basis for the mutagenicity of O 6 -methyl-guanine lesions

“…5). This methyl-proton:carbonyl interaction was not observed in previous crystallographic studies of B form DNA duplexes containing T⅐O6MeG pairs in the absence of protein (30). The BF polymerase binds DNA in an A form conformation with significant base pair opening (SI Table 4), providing additional space to accommodate the methyl group.…”

“…The O 6 -methyl group in O6MeG⅐T or O6MeG⅐C base pairs has been observed in ref. 30 or modeled in refs. 27 and 29 with the methyl group pointing away from the Watson-Crick face.…”

“…C⅐O6MeG pairs are more stable than the other three possible O6MeG pairings (but are weaker than canonical C⅐G pairings by 4-5 kcal/mol) (28). O6MeG⅐T mispairs are relatively unstable, forming only one hydrogen bond, but adopt a shape similar to Watson-Crick pairs (29,30) (Fig. 1b).…”

O6-Methylguanosine leads to position-dependent effects on ribosome speed and fidelity

“…Structures of a high-fidelity DNA polymerase in complex with m 6 G show that the incoming m 6 G:T base pair adopts a conformation that is nearly indistinguishable from canonical Watson-Crick base pairs (Warren et al 2006). Moreover, after replication, the m6G:T base pair is similar enough to G:C that it is not detected by the mismatch-repair machinery (Leonard et al 1990) but is instead detected by proteins devoted entirely to removing m 6 G lesions (Demple et al 1982). How could the m 6 G:T base pair trick the DNA replication machinery and yet m 6 G:U at the second codon position of the A site stall the ribosome?…”

Crystal structures of nucleic acids and their drug complexes

“…The structures of two dodecanucleotide duplexes containing this mismatch have been reported. 55,56 The base pairing in both is analogous to the normal WatsonCrick arrangement (Fig. 3), whereas NMR solution studies 57 have suggested that the N1G· · ·N3T hydrogen bond is absent or very weak.…”