Dataset Information

Mutations at several loci cause increased expression of ribonucleotide reductase in Escherichia coli.

ABSTRACT: Production of deoxyribonucleotides for DNA synthesis is an essential and tightly regulated process. The class Ia ribonucleotide reductase (RNR), the product of the nrdAB genes, is required for aerobic growth of Escherichia coli. In catalyzing the reduction of ribonucleotides, two of the cysteines of RNR become oxidized, forming a disulfide bond. To regenerate active RNR, the cell uses thioredoxins and glutaredoxins to reduce the disulfide bond. Strains that lack thioredoxins 1 and 2 and glutaredoxin 1 do not grow because RNR remains in its oxidized, inactive form. However, suppressor mutations that lead to RNR overproduction allow glutaredoxin 3 to reduce sufficient RNR for growth of these mutant strains. We previously described suppressor mutations in the dnaA and dnaN genes that had such effects. Here we report the isolation of new mutations that lead to increased levels of RNR. These include mutations that were not known to influence production of RNR previously, such as a mutation in the hda gene and insertions in the nrdAB promoter region of insertion elements IS1 and IS5. Bioinformatic analysis raises the possibility that IS element insertion in this region represents an adaptive mechanism in nrdAB regulation in E. coli and closely related species. We also characterize mutations altering different amino acids in DnaA and DnaN from those isolated before.

SUBMITTER: Feeney MA

PROVIDER: S-EPMC3294829 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Mutations at several loci cause increased expression of ribonucleotide reductase in Escherichia coli.

Feeney Morgan Anne MA Ke Na N Beckwith Jon J

Journal of bacteriology 20120113 6

Production of deoxyribonucleotides for DNA synthesis is an essential and tightly regulated process. The class Ia ribonucleotide reductase (RNR), the product of the nrdAB genes, is required for aerobic growth of Escherichia coli. In catalyzing the reduction of ribonucleotides, two of the cysteines of RNR become oxidized, forming a disulfide bond. To regenerate active RNR, the cell uses thioredoxins and glutaredoxins to reduce the disulfide bond. Strains that lack thioredoxins 1 and 2 and glutared ...[more]

PMID: 22247510

Similar Datasets

Project description:Expression of the Escherichia coli dnaN-encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. A mutant clamp (βE202K bearing a glutamic acid-to-lysine substitution at residue 202) binds to DNA polymerase III (Pol III) with higher affinity than the wild-type clamp, suggesting that its failure to impede growth is independent of its ability to sequester Pol III away from the replication fork. Our results demonstrate that the dnaNE202K strain underinitiates DNA replication due to insufficient levels of DnaA-ATP and expresses several DnaA-regulated genes at altered levels, including nrdAB, that encode the class 1a ribonucleotide reductase (RNR). Elevated expression of nrdAB was dependent on hda function. As the β clamp-Hda complex regulates the activity of DnaA by stimulating its intrinsic ATPase activity, this finding suggests that the dnaNE202K allele supports an elevated level of Hda activity in vivo compared with the wild-type strain. In contrast, using an in vitro assay reconstituted with purified components the βE202K and wild-type clamp proteins supported comparable levels of Hda activity. Nevertheless, co-overexpression of the nrdAB-encoded RNR relieved the growth defect caused by elevated levels of the β clamp. These results support a model in which increased cellular levels of DNA precursors relieve the ability of elevated β clamp levels to impede growth and suggest either that multiple effects stemming from the dnaNE202K mutation contribute to elevated nrdAB levels or that Hda plays a noncatalytic role in regulating DnaA-ATP by sequestering it to reduce its availability. IMPORTANCE DnaA bound to ATP acts in initiation of DNA replication and regulates the expression of several genes whose products act in DNA metabolism. The state of the ATP bound to DnaA is regulated in part by the β clamp-Hda complex. The dnaNE202K allele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While the dnaNE202K strain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and βE202K clamp proteins support equivalent levels of Hda activity in vitro. Taken together, these results suggest that βE202K-Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed.

Dataset Information

Mutations at several loci cause increased expression of ribonucleotide reductase in Escherichia coli.

Publications

Mutations at several loci cause increased expression of ribonucleotide reductase in Escherichia coli.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets