Mechanistic Studies on E. coli DNA Polymerase III Holoenzyme at the Replication Fork

The E. coli chromosome is replicated by a dimeric DNA polymerase III holoenzyme (Pol III HE) in a reaction where continuous leading and discontinuous lagging strand synthesis are coupled. Two models have been proposed to depict how a lagging strand polymerase dissociates from the preceding Okazaki fragment and cycle to the next primer. The collision model proposes that the polymerase collides with the 5\u27-end of the preceding Okazaki fragment and triggers release, whereas the signaling model suggests that the polymerase is signaled to cycle by synthesis of a new primer by primase. I developed a mini-circle DNA replication system with a highly asymmetric G:C distribution between DNA strands to differentiate these models. Specific perturbations of lagging strand synthesis by incorporation of ddGTP (chain termination) or dGDPNP (decreased elongation rate) on dCMP-containing lagging strand template confirm the signaling model and rule out the collision model. The lagging strand polymerase elongates much faster than the leading strand polymerase, explaining why gaps between Okazaki fragments are not found under physiological conditions. The presence of a primer, not primase, provides the signal to trigger cycling. Full-length Okazaki fragments (in the presence of dNTPs) and equivalent gaps between fragments (in the presence of dGDPNP) were obtained using reconstituted E. coli replicase regardless of the number of the τ DnaX subunits present in the clamp loader. I characterized an intrinsic helicase-independent strand displacement activity of the DNA Pol III HE and found that Pol III is stabilized by an interaction with SSB on the displaced strand by a Pol III-τ-ψ-χ-SSB interaction network. PriA, the initiator of replication restart on stalled replication forks, blocks the displacement reaction. E. coli SSB functions as a homotetramer with each subunit possessing a C-terminus interacting with other proteins that function in DNA replication and repair. To assess how many C-termini of SSB are required for function in DNA replication, I carried out rolling circle DNA replication assays using concatemeric forms of SSB that possess only one or two C-termini. I discovered that SSB \u22tetramers\u22 with one C-terminus cause a decrease in DNA synthesis and uncouples leading and lagging strand synthesis