Relationship between Fork Progression and Initiation of Chromosome Replication in E. coli

Ribonucleoside diphosphate reductase (RNR) of Escherichia coli is the prototype of the class I reductases common to most prokaryotes and eukaryotes from viruses to man. It is the only specific enzyme required, under aerobic growth, for the enzymatic formation of deoxyribonucleotides, the precursors for DNA synthesis. DNA replication requires a balanced supply of the four dNTPs, which explains the complex allosteric control of the enzyme (reviewed in Nordlund & Reichard, 2006). The active enzyme is a 1:1 complex of two subunits called proteins R1 and R2, each consisting of two polypeptide chains, coded by the genes nrdA and nrdB, respectively (Hanke & Fuchs, 1983). Although about 3000 nucleotides have to be consumed per second when a bacterium replicates its chromosome with two replication forks, only a very small pool of dNTP is accumulated in the cells. This pool would permit replication for no longer than half a minute (Werner, 1971; Pato, 1979). Channeling of the biosynthesis and compartmentation of the precursors has been proposed as explanations of how this shortage may be circumvented (Mathews, 1993; Kim et al., 2005) To satisfy the changing demand for the four deoxynucleotides, RNR must be closely associated with the replication machinery. In the aforementioned studies, Mathews et al., found evidence for the association of this enzyme with others related to the precursor biosynthesis, and coined the term dNTP-synthesizing complex (Mathews, 1993). The best-known defective RNR mutant in E. coli contains a thermolabile R1 subunit, coded by the nrdA101 allele. This allele carries a missense mutation, causing a change in amino acid 89 (L89P) (Odsbu et al., 2009). This leucine-to-proline substitution is close to the ATP cone domain that is located in the N-terminal region of the R1 protein and is, according to the holoenzyme model, located close to the R1-R2 interaction surface (Uhlin & Eklund, 1994), although no structural analysis of the mutant protein has been performed.