Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli: Ribonucleoside diphosphate reductase in the replication complex (original) (raw)
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BMC Molecular Biology, 2010
Background: There has long been evidence supporting the idea that RNR and the dNTP-synthesizing complex must be closely linked to the replication complex or replisome. We contributed to this body of evidence in proposing the hypothesis of the replication hyperstructure. A recently published work called this postulate into question, reporting that NrdB is evenly distributed throughout the cytoplasm. Consequently we were interested in the localization of RNR protein and its relationship with other replication proteins. Results: We tagged NrdB protein with 3×FLAG epitope and detected its subcellular location by immunofluorescence microscopy. We found that this protein is located in nucleoid-associated clusters, that the number of foci correlates with the number of replication forks at any cell age, and that after the replication process ends the number of cells containing NrdB foci decreases. We show that the number of NrdB foci is very similar to the number of SeqA, DnaB, and DnaX foci, both in the whole culture and in different cell cycle periods. In addition, interfoci distances between NrdB and three replication proteins are similar to the distances between two replication protein foci. Conclusions: NrdB is present in nucleoid-associated clusters during the replication period. These clusters disappear after replication ends. The number of these clusters is closely related to the number of replication forks and the number of three replication protein clusters in any cell cycle period. Therefore we conclude that NrdB protein, and most likely RNR protein, is closely linked to the replication proteins or replisome at the replication fork. These results clearly support the replication hyperstructure model.
Molecular Microbiology, 2002
Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli of 70 bp s-1. In contrast to this difference in polymerization, the dNTP pool is about 10 times smaller than the NTP pool (Pato, 1979). This discrepancy was observed very early on by Werner (1971), who asked how the intracellular concentration of dNTP could be sufficient to support the observed rate of DNA replication. Besides this difference in pools, dNTPs are highly specialized molecules, as they have few roles outside DNA replication, and this functionality is highly localized at only a few intracellular sites. In a work on the isolation of a DNA replication system bound to membrane in rat liver and hepatomes, Baril et al. (1974) demonstrated the incorporation of thymidine in their in vitro system and were the first to propose a multienzyme replication complex in which DNA polymerase II and at least three enzymes involved in the dNTP biosynthesis take part. Since then, many experiments have demonstrated the presence of some of the enzymes involved in dNTP synthesis in a multienzyme complex in both prokaryotic and eukaryotic cells (reviewed by Mathews, 1993). Three observations suggest a multienzyme complex for dNTP biosynthesis associated with the DNA replication apparatus: (i) the incorporation of radiolabelled thymidine into DNA reaches its maximal rate before the pool of dTTP is fully labelled (Werner, 1971; Pato, 1979); (ii) permeabilized bacterial cells incorporate deoxyribonucleoside diphosphates into DNA more efficiently than the corresponding triphosphates; and (iii) inhibition of nucleoside diphosphate kinase inhibits direct incorporation of dNTP into DNA in permeabilized cells (Reddy and Mathews, 1978). This model of a multienzyme complex also suggests that the transfer of dNTP to DNA polymerase is facilitated by channelling and com
Induction of Chromosome Replication as a Heat Stress Response inEscherichia coli
Modern Multidisciplinary Applied Microbiology, 2006
An upshift in the growth temperature of an E. coli culture causes induction of extra rounds of chromosome replication. This heat-induced replication (HIR) initiates at oriC, is transitory, and requires RNase H1 and RecA proteins but neither RNA polymerase activity nor de novo protein synthesis. HIR is not induced by the heat shock response and cannot be considered as SDR but as a recombinationdependent replication. It is SOS induction-independent, and needs RecA homologous recombinase and structural stabilization activities in a RecD exonuclease-dependent way, and RecBCD helicase function. DSBs are not generated during HIR, and possibly no D-loop structures. We suggest a structural stabilization of a heat opened structure in oriC via RecA and RecBCD, although other implications in the maintenance of replications forks during HIR elongation cannot be excluded.
Microbiology, 2011
Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of deoxyribonucleotides (dNTPs). Surprisingly, Escherichia coli cells carrying the nrdA101 allele, which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 6C under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at 42 6C and that its degradation under restrictive conditions is prevented by the presence of rifampicin. Nevertheless, the mere stability of the RNR protein at 42 6C cannot explain the completion of chromosomal DNA replication in the nrdA101 mutant. We found that inactivation of the DnaA protein by using several dnaAts alleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in the nrdA101 mutant at 42 6C. As both inactivation of the DnaA protein and inhibition of RNA synthesis block the occurrence of new DNA initiations, the consequent decrease in the number of forks per chromosome could be related to those effects. In support of this notion, we found that avoiding multifork replication rounds by the presence of moderate extra copies of datA sequence increases the relative amount of DNA synthesis of the nrdA101 mutant at 42 6C. We propose that a lower replication fork density results in an improvement of the progression of DNA replication, allowing replication of the entire chromosome at the restrictive temperature. The mechanism related to this effect is also discussed.
Journal of Bacteriology, 2011
Cells carrying the thermosensitive nrdA101 allele are able to replicate entire chromosomes at 42°C when new DNA initiation events are inhibited. We investigated the role of the recombination enzymes on the progression of the DNA replication forks in the nrdA101 mutant at 42°C in the presence of rifampin. Using pulsed-field gel electrophoresis (PFGE), we demonstrated that the replication forks stalled and reversed during the replication progression under this restrictive condition. DNA labeling and flow cytometry experiments supported this finding as the deleterious effects found in the RecB-deficient background were suppressed specifically by the absence of RuvABC; however, this did not occur in a RecG-deficient background. Furthermore, we show that the RecA protein is absolutely required for DNA replication in the nrdA101 mutant at restrictive temperature when the replication forks are reversed. The detrimental effect of the recA deletion is not related to the chromosomal degradation caused by the absence of RecA. The inhibition of DNA replication observed in the nrdA101 recA mutant at 42°C in the presence of rifampin was reverted by the presence of the wild-type RecA protein expressed ectopically but only partially suppressed by the RecA protein with an S25P mutation [RecA(S25P)], deficient in the rescue of the stalled replication forks. We propose that RecA is required to maintain the integrity of the reversed forks in the nrdA101 mutant under certain restrictive conditions, supporting the relationship between DNA replication and recombination enzymes through the stabilization and repair of the stalled replication forks.
Cellular & Molecular Biology Letters, 2007
NDP reductase activity can be inhibited either by treatment with hydroxyurea or by incubation of an nrdA ts mutant strain at the non-permissive temperature. Both methods inhibit replication, but experiments on these two types of inhibition yielded very different results. The chemical treatment immediately inhibited DNA synthesis but did not affect the cell and nucleoid appearance, while the incubation of an nrdA101 mutant strain at the non-permissive temperature inhibited DNA synthesis after more than 50 min, and resulted in aberrant chromosome segregation, long filaments, and a high frequency of anucleate cells. These phenotypes are not induced by SOS. In view of these results, we suggest there is an indirect relationship between NDP reductase and the chromosome segregation machinery through the maintenance of the proposed replication hyperstructure.
Microbiology, 2011
Ribonucleoside diphosphate reductase (RNR) is located in discrete foci in a number that increases with the overlapping of replication cycles in Escherichia coli. Comparison of the numbers of RNR, DnaX and SeqA protein foci with the number of replication forks at different growth rates reveals that fork : focus ratios augment with increasing growth rates, suggesting a higher cohesion of the three protein foci with increasing number of forks per cell. Quantification of NrdB and SeqA proteins per cell showed: (i) a higher amount of RNR per focus at faster growth rates, which sustains the higher cohesion of RNR foci with higher numbers of forks per cell; and (ii) an equivalent amount of RNR per replication fork, independent of the number of the latter.
Defects in ribosome function delay the initiation of chromosome replication in Escherichia coli
Journal of basic microbiology, 2018
The Sra protein is a component of the 30S ribosomal subunit while RimJ is a ribosome-associated protein that plays a role in the maturation of the 30S ribosomal subunit. Here we found that Δsra and ΔrimJ cells showed a delayed initiation of DNA replication, prolonged doubling time, decreased cell size, and decreased amounts of total protein and DnaA per cell compared with these observed for wild-type cells. A temperature sensitivity test demonstrated that absence of the Sra or RimJ protein did not change the temperature sensitivity of the dnaA46, dnaB252, or dnaC2 mutants. Moreover, ectopic expression of Sra reversed the mutant phenotype while cells carrying the pACYC177-rimJ plasmid did not reverse the rimJ mutant phenotype. The results indicate that deletion of sra or rimJ cause defects in ribosomal function and affect the translation process, leading to a decrease in synthesis of proteins including DnaA. Therefore, we conclude that Sra- and RimJ-mediated ribosomal function is req...