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 suhtilis

摘要

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 withthe 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 laggingstrand-specific subcomplex comprising DnaG, DnaE_(Bs) and DnaC, which stimulates chromosomal replication with enhanced fidelity.