Functional interplay of DnaE polymerase,DnaG primase and DnaC helicase within a ternary complex,and primase to polymerase hand-off during lagging strand DNA replication in Bacillus subtilis

Bacillus subtilis has two replicative DNA polymerases. PolC is a processive high-fidelity replicative polymerase, while the error-prone DnaE_Bs extends RNA primers before hand-off to PolC at the lagging strand. We show that DnaE_Bs interacts with the replicative helicase DnaC and primase DnaG in a ternary complex. We characterize their activities and analyse the functional significance of their interactions using primase, helicase and primer extension assays, and a ‘stripped down’ reconstituted coupled assay to investigate the coordinated displacement of the parental duplex DNA at a replication fork, synthesis of RNA primers along the lagging strand and hand-off to DnaE_Bs. The DnaG–DnaE_Bs hand-off takes place after de novo polymerization of only two ribonucleotides by DnaG, and does not require other replication proteins. Furthermore, the fidelity of DnaE_Bs is improved by DnaC and DnaG, likely via allosteric effects induced by direct protein–protein interactions that lower the efficiency of nucleotide mis-incorporations and/or the efficiency of extension of mis-aligned primers in the catalytic site of DnaE_Bs. We conclude that de novo RNA primer synthesis by DnaG and initial primer extension by DnaE_Bs are carried out by a lagging strand–specific subcomplex comprising DnaG, DnaE_Bs and DnaC, which stimulates chromosomal replication with enhanced fidelity.