LAUSR

Abstract Family A DNA polymerases (PolAs) form an important and well-studied class of extant polymerases participating in DNA replication and repair. Nonetheless, despite the characterization of multiple subfamilies in independent, dedicated works, their comprehensive classification thus far is missing. We therefore re-examine all presently available PolA sequences, converting their pairwise similarities into positions in Euclidean space, separating them into 19 major clusters. While 11 of them correspond to known subfamilies, eight had not been characterized before. For every group, we compile their general characteristics, examine their phylogenetic relationships and perform conservation analysis in the essential sequence motifs. While most subfamilies are linked to a particular domain of life (including phages), one subfamily appears in Bacteria, Archaea and Eukaryota. We also show that two new bacterial subfamilies contain functional enzymes. We use AlphaFold2 to generate high-confidence prediction models for all clusters lacking an experimentally determined structure. We identify new, conserved features involving structural alterations, ordered insertions and an apparent structural incorporation of a uracil-DNA glycosylase (UDG) domain. Finally, genetic and structural analyses of a subset of T7-like phages indicate a splitting of the 3′–5′ exo and pol domains into two separate genes, observed in PolAs for the first time.