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Suppression of a DnaX temperature-sensitive polymerization defect by mutation in the initiation gene, dnaA, requires
A Blinkova1, E Ginés-Candelaria, J D Ross
1Section of Molecular Genetics and Microbiology, and Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712, USA.
Abstract:
Temperature sensitivity of DNA polymerization and growth, resulting from mutation of the tau and gamma subunits of Escherichia coli DNA polymerase III, are suppressed by Cs,Sx mutations of the initiator gene, dnaA. These mutations simultaneously cause defective initiation at 20 degrees C. Efficient suppression, defined as restoration of normal growth rate at 39 degrees C to essentially all the cells, depends on functional oriC. Increasing DnaA activity in a strain capable of suppression, by introducing a copy of the wild-type allele, increasing the suppressor gene dosage or introducing a seqA mutation, reversed the suppression. This suggests that the suppression mechanism depends on reduced activity of DnaACs, Sx. Models that assume that suppression results from an initiation defect or from DnaACs,Sx interaction with polymerization proteins during nascent strand synthesis are proposed.
Insights
Mutations in Escherichia coli DNA polymerase III subunits affecting DNA growth are suppressed by dnaA mutations. These dnaA mutations also impair DNA initiation, suggesting a link between initiation and DNA polymerization.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- Escherichia coli DNA polymerase III (Pol III) is essential for DNA replication.
- Mutations in the tau and gamma subunits of Pol III cause temperature sensitivity in DNA polymerization and cell growth.
- The initiator gene, dnaA, plays a crucial role in initiating DNA replication.
Purpose of the Study:
- To investigate the relationship between DNA polymerization, initiation, and temperature sensitivity in Escherichia coli.
- To elucidate the mechanism by which dnaA mutations suppress temperature sensitivity caused by Pol III subunit mutations.
- To explore the role of DnaA protein activity in DNA replication fidelity and cell growth.
Main Methods:
- Genetic analysis of Escherichia coli strains with mutations in DNA polymerase III subunits and the dnaA gene.
- Assessment of cell growth rates at different temperatures (20°C and 39°C).
- Evaluation of the dependence of suppression on functional oriC (origin of replication).
- Manipulation of DnaA protein activity through gene dosage and additional mutations (e.g., seqA).
Main Results:
- Cs,Sx mutations in dnaA suppressed the temperature sensitivity of DNA polymerization and growth caused by tau and gamma subunit mutations.
- These dnaA mutations also resulted in defective DNA initiation at 20°C.
- Efficient suppression required a functional oriC.
- Increased DnaA activity (wild-type allele, increased gene dosage, or seqA mutation) reversed the suppression.
- Suppression was linked to reduced activity of DnaA protein.
Conclusions:
- The suppression mechanism involves reduced activity of the DnaA initiator protein.
- The findings suggest a potential interaction between DnaA protein and DNA polymerization machinery.
- Two models are proposed: suppression via an initiation defect or via DnaA interaction with nascent strand synthesis proteins.