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The Alarmone (p)ppGpp Regulates Primer Extension by Bacterial Primase
Christina N Giramma1, McKenna B DeFoer1, Jue D Wang1
1Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA.
The bacterial enzyme primase, crucial for DNA replication, is regulated by (p)ppGpp during starvation. This molecule inhibits primer extension and protects DNA replication fidelity under stress.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Primase synthesizes RNA primers essential for DNA replication initiation.
- Bacterial primase activity, specifically in Bacillus subtilis, is regulated by the alarmone (p)ppGpp.
- This regulation is vital for inhibiting DNA replication during amino acid starvation.
Purpose of the Study:
- To characterize the in vitro effects of (p)ppGpp on Bacillus subtilis DnaG primase activity.
- To dissect the influence of ppGpp on primer initiation, extension, and fidelity.
- To elucidate the combined impact of (p)ppGpp and GTP levels on primase function.
Main Methods:
- Utilized a single-nucleotide resolution primase assay.
- Investigated the impact of varying ppGpp and GTP concentrations.
- Analyzed primer extension, processivity, and base-pairing fidelity.
Main Results:
- ppGpp mildly affects primer initiation but strongly inhibits primer extension and reduces primase processivity.
- High (p)ppGpp and low GTP concentrations additively inhibit primase activity.
- ppGpp protects priming fidelity by reducing readthrough, even at low GTP concentrations.
Conclusions:
- ppGpp plays a critical role in regulating DNA replication during starvation by inhibiting primer extension.
- The combined effect of high (p)ppGpp and low GTP explains replication elongation inhibition in B. subtilis.
- ppGpp safeguards replisome integrity and genome stability under fluctuating nucleotide conditions and environmental stress.
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