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Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function
1Biochemistry and Structural Biology Graduate Program, Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA.
Abstract:
Escherichia coli strains lacking PriA are severely compromised in their ability to repair UV-damaged DNA and to perform homologous recombination. These phenotypes arise because of a lack of PriA-directed replication fork assembly at recombination intermediates such as D-loops. Naturally arising suppressor mutations in dnaC restore strains carrying the priA2::kan null allele to wild-type function. We have cloned one such gene, dnaC810, and overexpressed, purified, and characterized the DnaC810 protein. DnaC810 can support a PriA-independent synthesis of phiX174 complementary strand DNA. This can be attributed to its ability, unlike wild-type DnaC, to catalyze a SSB-insensitive general priming reaction with DnaB and DnaG on any SSB-coated single-stranded DNA. Gel mobility shift analysis revealed that DnaC810 could load DnaB directly to SSB-coated single-stranded DNA as well as to D loop DNA. This explains the ability of DnaC810 to bypass the requirement for PriA, PriB, PriC, and DnaT during replication fork assembly at recombination intermediates.
Insights
Mutant DnaC810 protein bypasses the need for PriA in DNA repair and recombination by independently loading DnaB onto DNA. This restores function in Escherichia coli lacking PriA, aiding UV DNA repair and homologous recombination.
Area of Science:
- Molecular Biology
- DNA Replication and Repair
- Bacteriology
Background:
- PriA protein is essential for DNA repair and homologous recombination in Escherichia coli.
- Loss of PriA function impairs replication fork assembly at recombination intermediates like D-loops.
- Suppressor mutations in dnaC can restore function to priA null mutants.
Purpose of the Study:
- To characterize the DnaC810 protein, a suppressor mutation of dnaC.
- To elucidate the mechanism by which DnaC810 bypasses the requirement for PriA.
- To understand the role of DnaC810 in replication fork assembly.
Main Methods:
- Cloning and overexpression of the dnaC810 gene.
- Purification and biochemical characterization of the DnaC810 protein.
- In vitro assays for DNA synthesis and protein-DNA interactions (gel mobility shift).
Main Results:
- DnaC810 protein supports PriA-independent synthesis of phiX174 complementary strand DNA.
- DnaC810 catalyzes a single-stranded DNA-binding protein (SSB)-insensitive priming reaction with DnaB and DnaG.
- DnaC810 directly loads DnaB onto SSB-coated single-stranded DNA and D-loop DNA.
Conclusions:
- DnaC810 protein bypasses the requirement for PriA, PriB, PriC, and DnaT in replication fork assembly at recombination intermediates.
- The unique priming activity of DnaC810 explains its ability to restore function in priA mutants.
- This finding provides insights into alternative pathways for replication restart and DNA repair.