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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.

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.

SUBMITTER: Babu VMP

PROVIDER: S-EPMC8570271 | biostudies-literature | 2021 Nov

REPOSITORIES: biostudies-literature

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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.

Babu Vignesh M P VMP Homiski Caleb C Scotland Michelle K MK Chodavarapu Sundari S Kaguni Jon M JM Sutton Mark D MD

Journal of bacteriology 20210920 23

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 demonstr ...[more]

PMID: 34543109

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Project description:Ribonucleotide reductases (RRs) are evolutionarily-conserved enzymes that catalyze the rate-limiting step during dNTP synthesis in mammals. RR consists of both large (R1) and small (R2) subunits, which are both required for catalysis by the R1(2)R2(2) heterotetrameric complex. Poxviruses also encode RR proteins, but while the Orthopoxviruses infecting humans [e.g. vaccinia (VACV), variola, cowpox, and monkeypox viruses] encode both R1 and R2 subunits, the vast majority of Chordopoxviruses encode only R2 subunits. Using plaque morphology, growth curve, and mouse model studies, we investigated the requirement of VACV R1 (I4) and R2 (F4) subunits for replication and pathogenesis using a panel of mutant viruses in which one or more viral RR genes had been inactivated. Surprisingly, VACV F4, but not I4, was required for efficient replication in culture and virulence in mice. The growth defects of VACV strains lacking F4 could be complemented by genes encoding other Chordopoxvirus R2 subunits, suggesting conservation of function between poxvirus R2 proteins. Expression of F4 proteins encoding a point mutation predicted to inactivate RR activity but still allow for interaction with R1 subunits, caused a dominant negative phenotype in growth experiments in the presence or absence of I4. Co-immunoprecipitation studies showed that F4 (as well as other Chordopoxvirus R2 subunits) form hybrid complexes with cellular R1 subunits. Mutant F4 proteins that are unable to interact with host R1 subunits failed to rescue the replication defect of strains lacking F4, suggesting that F4-host R1 complex formation is critical for VACV replication. Our results suggest that poxvirus R2 subunits form functional complexes with host R1 subunits to provide sufficient dNTPs for viral replication. Our results also suggest that R2-deficient poxviruses may be selective oncolytic agents and our bioinformatic analyses provide insights into how poxvirus nucleotide metabolism proteins may have influenced the base composition of these pathogens.

Dataset Information

Elevated Levels of the Escherichia coli nrdAB-Encoded Ribonucleotide Reductase Counteract the Toxicity Caused by an Increased Abundance of the β Clamp.

Publications

Elevated Levels of the Escherichia coli <i>nrdAB</i>-Encoded Ribonucleotide Reductase Counteract the Toxicity Caused by an Increased Abundance of the β Clamp.

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