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. (C) 2008 Elsevier Ltd. All rights reserved.