Regulation of Bacterial DNA Replication At the Level of Initiation

In all organisms, as well as autonomously replicating DNA molecules of organelles and plasmids, replication is divided into three stages: initiation, chain elongation, and termination. The control of replication occurs primarily at the level of initiation of DNA synthesis at the “origin” (ori site). Because DNA chains cannot be started de novo and requires a primer, the initiation complex contains primase activity for synthesis of an RNA primer. Discontinuous synthesis of Okazaki fragments needs repeated primer synthesis for each fragment as an integral component of chain elongation. Initiation of the primer at the ori sequence rather than elongation of initiated chains is the critical event in DNA replication control.

Different replicons of prokaryotes and eukaryotes utilize distinct mechanisms which vary in complexity, depending on the complexity of the organisms. A common feature of replication initiation control in E. coli genomes and plasmids is the presence of repeats of AT rich sequences which facilitate unwinding of DNA and one or multiple repeats of a “dnaA box” to which the initiator DnaA protein in E. coli or its functional homolog (called Rep in other cases) binds to allow helical unwinding and primer synthesis. The level of DnaA protein regulates the initiation frequency and, in turn, is controlled at the level of transcription of the dnaA gene. Thus, there are complex negative autofeedback loops to control dnaA gene expression. DnaA regulates its own gene, and its steadystate level in the cell is determined by the cellular growth state. The frequency of replicon firing is dependent on the growth rate of the bacteria. As mentioned before, rapidly growing cells can have multiple copies of the genome, while cells with a very lowgrowth rate have only one copy. Furthermore, as expected in cells with multiple genome copies, the genes near the origin will have a higher average copy number than the genes located near the terminus of replication and, therefore, will be more transcriptionally active.

In the case of multicopy plasmids, the control of copy number is mediated by the synthesis of anti-sense RNA of the replication initiator protein Rep, which is copied from the nontranscribed DNA strand and is thus complementary to the normal RNA. Anti-sense RNA prevents synthesis of the Rep protein, which is required for initiation of DNA synthesis and whose concentration is the primary mechanism of controlling initiation frequency. Rep proteins encoded by plasmids bind to additional copies of binding sites called “iterons,” often present upstream of the ori sequences in the plasmids.