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Published on: August 21, 2016
Escherichia coli K-12 has two distinguishable PriA-PriB replication restart pathways
Steven J Sandler1, Maxime Leroux1,2, Tricia A Windgassen3,4
1Department of Microbiology, University of Massachusetts at Amherst, Amherst, Massachusetts, USA.
Insights
The study reveals two distinct PriA-PriB replication restart pathways in E. coli. One pathway relies on PriA
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA replication restart is essential for cell viability.
- In Escherichia coli, the PriA protein is central to multiple replication restart pathways.
- Understanding PriA's functions is key to comprehending DNA repair mechanisms.
Purpose of the Study:
- To investigate the distinct roles of PriA protein variants in DNA replication restart pathways.
- To biochemically characterize the ATPase and helicase activities of PriA mutants.
- To elucidate the genetic and biochemical basis of two separate PriA-PriB pathways.
Main Methods:
- Site-directed mutagenesis of the priA gene in Escherichia coli.
- Biochemical assays including ATPase and helicase activity measurements.
- In vivo phenotypic analysis of priA mutant strains.
- In vitro interaction studies with PriB and DNA.
Main Results:
- PriA300 mutant lacks ATPase and helicase activity but retains PriB interaction.
- PriA301 mutant shows reduced DNA and PriB interactions but retains helicase activity.
- A double mutant (PriA300,301) retains only DNA interaction ability.
- Mutant phenotypes suggest two distinct PriA-PriB pathways: one helicase-dependent, one helicase-independent.
Conclusions:
- The PriA protein utilizes distinct mechanisms in different replication restart pathways.
- Two biochemically and genetically separable PriA-PriB pathways exist.
- One pathway depends on PriA's helicase activity, while the other involves helicase-independent remodeling.
Abstract:
In Escherichia coli, PriA, PriB, PriC, and DnaT proteins mediate three pathways for Replication Restart called PriA-PriB, PriA-PriC, and PriC. PriA is crucial for two of the three pathways. Its absence leads to slow growth, high basal levels of SOS expression, poorly partitioning nucleoids, UV sensitivity, and recombination deficiency. PriA has ATPase and helicase activities and interacts with PriB, DnaT, and single-stranded DNA-binding protein (SSB). priA300 (K230R) and priA301 (C479Y) have no phenotype as single mutants, but each phenocopy a priA-null mutant combined with ∆priB. This suggested that the two priA mutations affected the helicase activity that is required for the PriA-PriC pathway. To further test this, the biochemical activities of purified PriA300 and PriA301 were examined. As expected, PriA300 lacks ATPase and helicase activities but retains the ability to interact with PriB. PriA301, however, retains significant PriB-stimulated helicase activity even though PriA301 interactions with PriB and DNA are weakened. A PriA300,301 variant retains only the ability to interact with DNA in vitro and phenocopies the priA-null phenotype in vivo. This suggests that there are two biochemically and genetically distinct PriA-PriB pathways. One uses PriB-stimulated helicase activity to free a region of ssDNA and the other uses helicase-independent remodeling activity.
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