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Link to DOI – https://doi.org/10.1101/2025.03.26.645540
Replicative DNA polymerases (DNAP) play a critical role in genome duplication, ensuring the accurate transmission of genetic information in all kingdoms of life. This process is essential also for DNA-dependent viruses, including bacteriophages. In many phage genomes, a uracil-DNA glycosylase (UDG) is encoded in trans. In this paper, we identify a new subfamily of A-family DNAP in phages that are fused to an active (UDG) domain. Two members of this subfamily, B. subtilis phage SP-15 and YerA41 are known to be hypermodified on their thymidines. Here, we present cryo-EM structures at high resolution for two of its members in various functional and conformational states, from YerA41 and phiLo phages. The structures explain how these DNAPs can have an activity, distinct from copying genetic information, which reads dU bases ahead of the replication fork and creates abasic sites that are efficiently bypassed by the DNAP. Additionally, we report the co-occurrence of both a X-family DNAP and a DNA ligase in the corresponding phage genomes, and show that both enzymes are capable of repairing the abasic sites. The former makes a stable complex with the replicative DNAP, which thus appears as a platform for recruitment of the enzymatic activities necessary for the repair of dU bases during replication, that result from the incorporation of residual dUTP in the pool of nucleotides. Strikingly, the location of the UDG domain is the same as in the structure of Mpox virus replicative complex involving a B-family DNAP.