A bacteriophage split DNA polymerase incorporates a sliding clamp-derived processivity subunit

Viruses that infect bacteria, termed bacteriophages or phages, typically encode their own DNA replication machinery that rapidly generates many copies of the phage genomic DNA in an infected cell. Nucleus-forming phages of the chimallivirus family possess a distinctive repertoire of predicted DNA replication proteins, with two proteins annotated as DNA polymerases plus a DnaB-like replicative helicase, but lacking an identified primase or sliding clamp protein. Here, we demonstrate that in the Pseudomonas aeruginosa -infecting chimallivirus PhiKZ, the core DNA polymerase comprises a stable 1:1 complex of two DNA polymerase subunits, termed DNAP ^N and DNAP ^C . A 2.1 Å resolution cryoelectron microscopy structure of this core complex reveals homology to other phage and bacterial B-family DNA polymerases. Next, we identify a direct but transient interaction between the core DNA polymerase complex and a third protein termed Rhc, which possesses an N-terminal RNase H domain and a C-terminal domain related to DNA sliding clamp proteins. Cryoelectron microscopy structures of a stabilized DNA polymerase-Rhc complex, and of this complex bound to a primer-template DNA, reveal a single Rhc subunit that cooperates with the DNA polymerase thumb domain to bind double-stranded DNA emerging from the DNA polymerase active site. While the Rhc RNase H domain is not resolved in our structures, this domain is situated such that it could degrade RNA associated with an incoming template DNA strand. In vitro primer-extension assays show that Rhc strongly boosts DNA polymerase activity, and that this boost relies on the Rhc-DNA polymerase interface we observe in our structures. Overall, our findings reveal a distinctive chimallivirus-specific DNA replication machine with an integrated DNA-binding subunit related to sliding clamp proteins, and a flexibly-linked RNase H domain.