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.

Fig. 1.

Inferred protonation states of C•A base pairs observed in the structures. In their canonical tautomeric state C and A do not pair (middle), because the two extracyclic amines clash. If either C or A tautomerizes (asterisk), a hydrogen-bonded base pair that mimicks a cognate shape can form (top). If A ionizes, a wobble base pair can form (bottom) (17, 18). Hydrogen bond donors and acceptors are colored blue and pink respectively.

Fig. 2.

DNA polymerase replication fidelity filters. Shaded areas correspond to fidelity filters: preinsertion site (n, orange), insertion site (n, blue), catalytic site (magenta), postinsertion site (n-1, pink), and DNA duplex-binding region (n-2 to n-5, gray). DNA primer (copper) and template strands (orange) are also shown. The O helix transitions from an open (magenta) through an ajar (gray) to a closed (blue) conformation. Cognate-shaped base pairs (blue) are positioned for catalysis in the closed state. Noncanonical shapes (gray) tend to be selected against in the ajar conformation. The polymerase makes hydrogen bonds with the minor groove of base pairs positioned at sites n-1 to n-5 in the duplex-binding region following incorporation. This figure combines information derived from four structures: open (1L3U) (15), ajar (3HP6) (29), and closed (2HVI and 3EZ5) (34, 42).

Fig. 3.

Comparison of C•A mismatch and T•A cognate base pairs placed at the polymerase insertion site. (A), (B) The C•A wobble (green) and T•A base (gray) pair obtained in the presence of Mg²⁺. For the C•A wobble pair, the O helix adopts the ajar conformation (A), the triphosphate is distorted, and the catalytic site is incompletely assembled (B). (D), (E) The C•A cognate shape (magenta) obtained in the presence of Mn²⁺. Comparison with a T•A base pair shows that the O helix is closed (D), the triphosphate is undistorted, and the active site fully assembled (E). (C), (F) Two views of composite omit maps of the C•A base pair (contoured at 1.2σ, C•A wobble; contoured at 2σ, C•A cognate) (green, Mg²⁺; purple, Mn²⁺). The presence of Mn²⁺ is confirmed by anomalous difference map (red, contoured at 4σ). (G), (H) Superposition of C•A wobble and C•A cognate at two different views showing the structural differences between the wobble and cognate conformations of this mismatch. (I) Variations of minor groove angles of C•A mismatch structures (wb, wobble; m, cognate mimic) or average cognate, Watson-Crick base-pair structures () captured at five different positions. λ_primer and λ_template are defined as the angle between the glycosidic bond of primer or template nucleotide and a line between the C1′ atoms of the base pair. Complete tables of all nine base-pair parameters are included (Table S3). Analysis shown here is based on molecule 1 of the two molecules in the asymmetric unit; molecule 2 is described in Table S3. The capture of a nucleotide at the insertion site involves the use of dideoxy analogs (34). Additional structures were determined with a 2′-deoxycytidine triphosphate which confirms the results described here (Fig. S1).

Fig. 4.

A water mediated hydrogen bond encodes edge recognition of cognate base-pair shapes. (A) C•A cognate shape mimic (Mn²⁺, magenta); (B) C•A wobble (Mg²⁺, green); (C) T•A; (D) A•T; (E) G•C; (F) C•G (from previously published structure; PDB code, 2HVI) (34). Composite omit maps (gray) at 1.5σ (A), (C–E) and 1.2σ (B) are shown around the base pairs and the anchored water molecule. Dashed lines (black) indicate hydrogen bonds.

Fig. 5.

Comparison of C•A mismatch and T•A cognate base-pair structures in the duplex region. (A–C) The C•A wobble base pair captured at the postinsertion site (1.53 Å resolution) showing overall structure (A), minor-groove interactions (B), and composite omit map (contoured at 1.8σ) around the mismatch (C). The next template base is disordered. (D), (E) C•A adopts a near-cognate shape at the n-3 (D) position (1.65 Å resolution) and a wobble shape at the n-4 (E) position (1.65 Å resolution). At both positions, minor groove interactions are maintained. (F) The C•A wobble observed at the n-6 position (1.60 Å resolution) where there are no contacts between the duplex DNA and the polymerase.

Fig. 6.

Insertion sites of representative members of five DNA polymerase families. (A) Superposition of three members of the A-family DNA polymerases: BF (yellow; PDB code, 2HVI) (34), Thermus aquaticus DNA polymerase I large fragment (cyan; 3KTQ) (21), and T7 bacteriophage DNA polymerase (pink;1T7P) (28). The interactions between the water molecule and the base, and the three anchoring protein residues are conserved in all three complexes. (B) A member of the C-family DNA polymerase, Geobacillus kaustophilus DNA polymerase PolC (3F2B) (43). A water molecule makes similar interaction with the incoming nucleotide base which is coordinated by a single histidine instead of the anchoring side chains. (C) A member of the X-family DNA polymerase, human DNA polymerase beta (2FMP) (44). The incoming nucleotide is hydrogen-bonded directly to an asparagine side chain instead of a water. Similar interactions are also present in another member of the family, human DNA polymerase lambda (1XSN) (45). (D) A member of the Y-family DNA polymerase, Sulfolobus solfataricus DNA polymerase IV (Dpo4) (2AGQ) (46). The water molecule contacting the nucleotide base is coordinated by a tyrosine instead of three anchoring residues. Similar interactions are also present in another member of the family, human DNA polymerase iota (1ZET) (47) (reviewed in ref. 48). (E) A member of the B-family DNA polymerase, Enterobacteria phage RB69 DNA polymerase (3NCI) (49). The base-pair edges are read out by Van der Waals interactions only, perhaps augmented by weak electrostatic interactions mediated by the glycine and the two ring protons of tyrosine (49). Similar interactions are also present in another member of the family, Bacillus phage phi29 DNA polymerase (2PYJ) (22). The selection of the structure for a representative polymerase family was based on resolution of the ternary complex.