Reconstitution of the B. subtilis Replisome with 13 Proteins Including Two Distinct Replicases

Abstract

We have expressed and purified 13 proteins predicted to be required for B. subtilis DNA replication. When combined with a circular DNA template with a 5' unpaired flap, these proteins reconstitute replication of both the leading and lagging strands at the physiological rate. Consistent with the in vivo requirement for two DNA polymerase III replicases for B. subtilis chromosomal replication, both PolC and DnaE are required for reconstitution of the replication fork in vitro. Leading strand synthesis requires PolC plus ten proteins; lagging strand synthesis additionally requires primase and DnaE. DnaE does not serve as the lagging strand replicase, like DNA polymerase delta in eukaryotes, but instead functions like eukaryotic DNA polymerase alpha, adding a stretch of deoxynucleotides to the RNA primer before handoff to PolC. Primase equilibrates with the fork prior to synthesis of each Okazaki fragment, and its concentration controls the frequency of initiation and Okazaki fragment size.