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Purification and characterization of DnaC810, a primosomal protein capable of bypassing PriA function

L Xu1, K J Marians

  • 1Biochemistry and Structural Biology Graduate Program, Cornell University Graduate School of Medical Sciences, New York, New York 10021, USA.

The Journal of Biological Chemistry
|March 14, 2000
PubMed

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.

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