Interactions of the Bacillus subtilis DnaE polymerase with replisomal proteins modulate its activity and fidelity

Abstract

During Bacillus subtilis replication two replicative polymerases function at the replisome to collectively carry out genome replication. In a reconstituted in vitro replication assay, PolC is the main polymerase while the lagging strand DnaE polymerase briefly extends RNA primers synthesized by the primase DnaG prior to handing-off DNA synthesis to PolC. Here, we show in vivo that (i) the polymerase activity of DnaE is essential for both the initiation and elongation stages of DNA replication, (ii) its error rate varies inversely with PolC concentration, and (iii) its misincorporations are corrected by the mismatch repair system post-replication. We also found that the error rates in cells encoding mutator forms of both PolC and DnaE are significantly higher (up to 15-fold) than in PolC mutants. In vitro, we showed that (i) the polymerase activity of DnaE is considerably stimulated by DnaN, SSB and PolC, (ii) its error-prone activity is strongly inhibited by DnaN, and (iii) its errors are proofread by the 3′ > 5′ exonuclease activity of PolC in a stable template-DnaE–PolC complex. Collectively our data show that protein–protein interactions within the replisome modulate the activity and fidelity of DnaE, and confirm the prominent role of DnaE during B. subtilis replication.

Keywords

• dna replication
• dna polymerase
• single-strand binding protein
• dna polymerase clamp
• dna polymerase proofreading
• mismatch repair