# 60 Years of Studies into the Initiation of Chromosome Replication in Bacteria
## 1 Introduction  
[PERSON] and [PERSON] proposed the _Replicon Theory_ on the basis of observations that had accumulated until about 1962. These observations included, firstly, the demonstration by [PERSON] and [PERSON] that de novo protein synthesis is required for the initiation of DNA replication in _Escherichia coli_[1] and, secondly, the work on sexual conjugation in _E. coli_. The conjugation between _Hfr_ and _F_- strains entails the transfer of the chromosome starting from a genetically determined, unique point on the chromosome of _Hfr_. [PERSON] and [PERSON] made the assumption that the normal replication of the chromosome is similar to that occurring during conjugation. They postulated that replication starts from a unique site after a specific interaction between a factor (the initiator) and the start site (the origin of replication). After an oral presentation of their hypothesis in 1962, they published a note entitled the _\"Replicon Theory\"_ in 1963 [2]. Coincidentally, in the same year, two important results supporting their hypothesis were published: firstly, a picture of the chromosome of _E. coli_ undergoing replication [3], and, secondly, evidence from germinating _B. subtilis_ for the sequential duplication of the chromosome from a unique site [4]. In order to test the predictions of the _Replicon Theory_, two groups, one in Paris (at the Pasteur Institute) and the other in Cambridge (at the Laboratory of Molecular Biology), started to isolate mutants that failed to initiate DNA replication. Such mutations are lethal unless they are in the form of conditional mutations such as those that confer pH sensitivity or temperature sensitivity. Starting from an _E. coli_ thymine-requiring mutant in which DNA synthesis was easily shown by its incorporation of radioactive thymidine, they isolated more than 100 mutants that grew at 30 \({}^{\circ}\)C but not at 42 \({}^{\circ}\)C. Around 10 mutants that failed to synthesize DNA at 42 \({}^{\circ}\)C were classified into those in which incorporation stoppedimmediately after the temperature shift to 42 \({}^{\circ}\)C and those in which incorporation only stopped after a delay. Significantly, two of their mutants (_CRT-46_ and _-83_) arrested thymidine incorporation at 42 \({}^{\circ}\)C with similar kinetics to that occurring at a permissive temperature in the presence of an inhibitor of protein synthesis, chloramphenicol (which echoed the earlier finding about initiation's need for protein synthesis [1]). Moreover, these two mutants allowed lambda phage, after infection, to resume DNA synthesis at high temperatures, whereas the other eight mutants failed. This could be explained by the _Replicon Theory_, which proposes that each replicating unit possesses its own initiator. The fact that phage replication was independent of the replication of the host chromosome (in addition to differences in the kinetics of DNA synthesis) in _CRT-46_ and _-83_ supported the idea that these mutants were defective specifically in the initiation of chromosome replication [5]. Subsequently, these two mutants were mapped, and the mutated gene was called _dnaA_[6]. The _Replicon Theory_ postulated that the nature of the initiator is independent of the enzymes responsible for DNA chain elongation. In the rolling circle model for _pli_ X174 phage DNA replication, a nickase can be the initiator [7]. Accordingly, a _dnaA83_ culture was briefly irradiated with gamma rays, which introduced nicks into the chromosome (and, less frequently, double-strand breaks). This irradiation provoked a new round of DNA synthesis, even at non-permissive temperatures [8]. This result shows that the action of DnaA as an initiator is independent of the system responsible for polymerizing DNA.