Fig. 2: Engineering and characterization of hypermutagenic T7 DNAP variants.

a, AlphaFold3-predicted⁶⁷ structure of variant v8, highlighting key engineered modifications: v1 (S399T), thumb domain relaxation affecting template minor groove interactions; v2 (T523R), fingers domain modification proximal to active site; v4 (D5A, E7A, Y64C and F120L), exonuclease domain inactivation; v7–9, fusion with TadA-8e adenosine deaminase or TadDE general deaminase enabling concurrent DNA deamination during replication. b, Mutational frequencies of engineered T7 DNAP variants measured by a fluctuation assay using a phagemid-encoded chloramphenicol resistance gene containing an internal stop codon. Wild-type T7 DNAP (WT), mutant T7 DNAPs (v1–v4), T7 DNAP v2.4 fused to TadA-8e (v7), T7 DNAP v4 fused to TadA-8e (v8) and T7 DNAP v4 fused to TadDE (v9) are shown. Data are shown as mean ± s.d. of n = 3 independent experiments. c, Snapshots from molecular dynamics simulations of T7 DNAP (PDB: 1T7P): wild type (top) and T523R variant mutated in silico (bottom) with A–G nucleotide mismatch. Hydrogens are not shown. Distances are measured in Ångström. d, Mutational frequencies of T7 DNAP variants determined through Illumina NGS using a 39-kb BAC phagemid. Mismatches were corrected for sequencing errors by subtracting wild-type mismatches. Data points represent mismatches across 39 kb binned into 20 equal-width bins. e, Substitution frequencies and ratios of A:T → G:C and C:G → T:A transitions for v8 and v9 variants derived from NGS data. Data points represent substitution frequencies for the respective transition types across 39 kb binned into 20 equal-width bins (Illumina sequencing, mean coverage >14,000× per base pair). Centre line represents the mean. f, Mutational spectra heat maps of LySE v8 and v9 showing the frequency of each substitution type calculated as a percentage of the total number of mutations observed. Rows represent the reference base; columns represent the mutated base. The sum of all values in the matrix equals 100%. g, Distribution of LySE v8 and v9 substitution frequencies across a 39-kb BAC phagemid (mean coverage = 14,098× per base pair). Diagram in g created in BioRender; Fredens, J. https://biorender.com/jomazxk (2026).