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Elevated Levels of the Escherichia coli nrdAB -Encoded Ribonucleotide Reductase Counteract the Toxicity Caused by an Increased Abundance of the β Clamp
- Source :
- J Bacteriol
- Publication Year :
- 2021
- Publisher :
- American Society for Microbiology, 2021.
-
Abstract
- 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 (β(E202K) 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 dnaN(E202K) 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 dnaN(E202K) 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 β(E202K) 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 dnaN(E202K) 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 dnaN(E202K) allele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While the dnaN(E202K) strain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and β(E202K) clamp proteins support equivalent levels of Hda activity in vitro. Taken together, these results suggest that β(E202K)-Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed.
- Subjects :
- DNA Replication
Models, Molecular
Ribonucleoside Diphosphate Reductase
Protein Conformation
DNA polymerase
Microbiology
Gene Expression Regulation, Enzymologic
RNA polymerase III
chemistry.chemical_compound
Bacterial Proteins
Ribonucleotide Reductases
Escherichia coli
Transcriptional regulation
Molecular Biology
DNA Polymerase III
DNA clamp
biology
Escherichia coli Proteins
DNA replication
Gene Expression Regulation, Bacterial
DnaA
Cell biology
DNA-Binding Proteins
Ribonucleotide reductase
chemistry
biology.protein
DNA
Research Article
Subjects
Details
- ISSN :
- 10985530 and 00219193
- Volume :
- 203
- Database :
- OpenAIRE
- Journal :
- Journal of Bacteriology
- Accession number :
- edsair.doi.dedup.....90faaf899f9d3cdd5d69ee8cb47f9840