Functional requirements for heat induced genome amplification in Escherichia coli

A temperature shift-up induces extra rounds of fully replicated chromosomes in Escherichia coli and leads to an increase in DNA/mass ratio. In the present work we characterize the requirements for this heat-induced replication (HIR) with respect to replisome components, replication restart, and recombination functions. We found that HIR requires Klenow and 5′–3′ exonuclease activities from Pol I and Pol III, but does not require translesion synthesis polymerases. We also found that replication restart is PriA–PriB pathway dependent. The dnaC809 allele suppresses the dependency on PriA, confirming the requirement for primosome assembly, in which PriA helicase function is not required. Rep helicase and the replication-associated function of RecA were found to be essential. HIR has low recombination requirements, and no DSBs are generated by heat stress. We propose that the Pol I-dependent replisome in HIR, which gives a slow replication speed, is more unstable and disassembles more frequently than normal replisome. Rep and RecA would be required to stabilize HIR replication forks. Since neither D-loops nor R-loops support HIR restart, the PriA–PriB pathway must reload the replisome by a direct restart mechanism from a 3′-end of a nascent leading strand.