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...