Archaeal family B DNA polymerase facilitates lagging strand DNA replication in the <i>Thermococcales</i>

Replicative DNA synthesis is distributed among several DNA polymerases (DNAPs) in both Bacteria and Eukarya. The division of synthesis permits specialized activities of unique DNAPs to facilitate rapid replication in a continuous manner on the leading strand and discontinuous synthesis of the lagging strand with repetitive priming and maturation of Okazaki fragments. Many archaea encode two evolutionarily unrelated DNAPs, PolB and PolD, but how leading and lagging strand synthesis responsibilities are partitioned between each polymerase has yet to be resolved in vivo. In the model archaeon Thermococcus kodakarensis, Pol B is dispensable, demonstrating PolD can independently support replicative synthesis, but does not exclude the possibility that PolB normally participates in key aspects of DNA replication. Here, we combine phenotypic analyses of strains encoding variant DNAPs, high-throughput sequencing, and purified replication assays to establish that PolB actively facilitates lagging strand synthesis in vivo. Pol B outperforms PolD at strand displacement and Okazaki fragment maturation, demonstrating that specialized DNAPs share replicative synthesis duties in most Archaea. Our findings define a foundational division of labor between PolB and PolD in Archaea and establish the use of specialized replicative DNAP activities for strand-specific synthesis across all life.