Structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis

Abstract

Even though high-fidelity polymerases copy DNA with remarkable accuracy, some base-pair mismatches are incorporated at low frequency, leading to spontaneous mutagenesis. Using high-resolution X-ray crystallographic analysis of a DNA polymerase that catalyzes replication in crystals, we observe that a C•A mismatch can mimic the shape of cognate base pairs at the site of incorporation. This shape mimicry enables the mismatch to evade the error detection mechanisms of the polymerase, which would normally either prevent mismatch incorporation or promote its nucleolytic excision. Movement of a single proton on one of the mismatched bases alters the hydrogen-bonding pattern such that a base pair forms with an overall shape that is virtually indistinguishable from a canonical, Watson-Crick base pair in double-stranded DNA. These observations provide structural evidence for the rare tautomer hypothesis of spontaneous mutagenesis, a long-standing concept that has been difficult to demonstrate directly.crystal structure | replication fidelity | mispair | polymerase structure H igh-fidelity polymerases replicate double-stranded DNA with remarkable accuracy (1). Fidelity is achieved by a successive series of conformational changes and molecular recognition events encoded at different sites on the polymerase surface such that mismatches are either prevented from incorporating or are excised within a few nucleotides past their incorporation point (2-5). At the site of covalent incorporation, shape complementarity between the polymerase surface and the edges of correctly paired bases is the dominant mechanism that determines specificity (6, 7). Here, mismatched base pairs or lesions that do not conform to this stereochemical constraint misalign their incoming triphosphate moiety relative to the 3′ OH of the growing primer terminus, leading to rejection of the incorrect or damaged nucleotides (2-4). However, modified bases that maintain the stereochemistry of cognate base-pair edges are readily incorporated (6-8). Nevertheless, polymerases do incorporate mismatched nucleotide base pairs at low frequency, leading to spontaneous mutagenesis (1).The mechanism by which spontaneous replication errors occur has long been the subject of intense speculation. In their second paper on the structure of DNA, Watson and Crick recognized that tautomerization alters the hydrogen-bonding patterns and therefore could enable mismatches to assume the structure of canonical base pairs (9). This notion was elaborated in the rare tautomer hypothesis of spontaneous mutagenesis, which states that mutations arise through the formation of high-energy tautomers at low frequency (8, 10). However, it has been challenging to obtain direct structural evidence for this mechanism. In the absence of polymerase, mismatches do not adopt a canonical base-pair structure in DNA (5, 11). Recently, a T•G mismatch has been observed to adopt a canonical base-pair structure in a polymerase, due to an ionization event, demonstrating that noncanonical hydrogen-bonding patt...

In silico phylogenomics using complete genomes: a case study on the evolution of hominoids

“…Our model choice results together with the unimodally distributed Ti/Tv estimates for ALs (and AE loci as well) suggest that these loci largely evolved via a simple mechanism that gives rise to a transition bias (Wakeley 1996). This finding is significant because it provides evidence in support of the long-standing "rare tautomer hypothesis" by Watson and Crick (1953), which holds that spontaneous base substitutions, which are specifically transitions, arise via incorporation of rare base tautomers during DNA replication (Harris et al 2003;Wang et al 2011). …”

Dynamic basis for dG•dT misincorporation via tautomerization and ionization

“…Decades later, it is well established that the replicative and translational machineries form a tight grip around the WC geometry to discriminate against mismatches 2–5 . There is also evidence that both tautomeric 6–13 (Fig. 1b) and anionic 7–9,14,15 (Fig.…”

“…4a) is required for misincorporation following initial dNTP binding in a wobble conformation, it could provide a mechanism for lowering k pol incorrect relative to k pol correct . Indeed, prior studies have shown that DNA polymerases cannot undergo the necessary conformational changes needed for catalysis when dG•dT is in a wobble conformation 7 and all available structures of catalytically active polymerases with bound mismatches within the active site feature WC-like dG•dT or dA•dC geometries 6,7 . Similarly, WC-like rG•rU mismatches have been shown to form in the first and second codon positions of catalytically active ribosomes 9 , in which wobbles are typically rejected 5 , potentially helping to explain translational error hotspots 41 .…”

“…1b) and anionic 7–9,14,15 (Fig. 1c) WC-like mismatches can evade such fidelity checkpoints and give rise to replication 6,7 and translation errors 16 . Despite their centrality to the fidelity of information transfer in the central dogma of molecular biology, and growing evidence showing the involvement of spontaneous mutations in generating cancer causing mutations 17 , the very existence of these species and their contributions to replication and translation errors remain to be definitively established.…”

“…1). Protons are generally invisible to X-ray crystallography and cryo-EM 12 , and consequently it has not been possible to unambiguously resolve the identity of WC-like mismatches captured within active sites of polymerases 6,7,15,23 and the ribosome decoding site 9,24 . Moreover, WC-like mismatches are predicted to exist in rapid tautomeric (G enol •T/U⇌G•T enol /U enol ) 25,26 equilibria (Fig.…”