Biochemical characterization of Escherichia coli DnaC variants that alter DnaB helicase loading onto DNA

J Biol Chem. 2024 May;300(5):107275. doi: 10.1016/j.jbc.2024.107275. Epub 2024 Apr 6.


DNA replication in Escherichia coli starts with loading of the replicative helicase, DnaB, onto DNA. This reaction requires the DnaC loader protein, which forms a 6:6 complex with DnaB and opens a channel in the DnaB hexamer through which single-stranded DNA is thought to pass. During replication, replisomes frequently encounter DNA damage and nucleoprotein complexes that can lead to replication fork collapse. Such events require DnaB re-loading onto DNA to allow replication to continue. Replication restart proteins mediate this process by recruiting DnaB6/DnaC6 to abandoned DNA replication forks. Several dnaC mutations that bypass the requirement for replication restart proteins or that block replication restart have been identified in E. coli. To better understand how these DnaC variants function, we have purified and characterized the protein products of several such alleles. Unlike wild-type DnaC, three of the variants (DnaC 809, DnaC 809,820, and DnaC 811) can load DnaB onto replication forks bound by single-stranded DNA-binding protein. DnaC 809 can also load DnaB onto double-stranded DNA. These results suggest that structural changes in the variant DnaB6/DnaC6 complexes expand the range of DNA substrates that can be used for DnaB loading, obviating the need for the existing replication restart pathways. The protein product of dnaC1331, which phenocopies deletion of the priB replication restart gene, blocks loading through the major restart pathway in vitro. Overall, the results of our study highlight the utility of bacterial DnaC variants as tools for probing the regulatory mechanisms that govern replicative helicase loading.

Keywords: AAA+ ATPase; ATPase; DNA; DNA helicase; DNA helicase loader; DNA replication; DNA replication restart; DnaB; DnaC.

MeSH terms

  • DNA Replication*
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • DNA, Single-Stranded / genetics
  • DNA, Single-Stranded / metabolism
  • DnaB Helicases* / chemistry
  • DnaB Helicases* / genetics
  • DnaB Helicases* / metabolism
  • Escherichia coli Proteins* / chemistry
  • Escherichia coli Proteins* / genetics
  • Escherichia coli Proteins* / metabolism
  • Escherichia coli* / genetics
  • Escherichia coli* / metabolism
  • Mutation


  • Escherichia coli Proteins
  • DnaB Helicases
  • DnaC protein, E coli
  • dnaB protein, E coli
  • DNA, Bacterial
  • DNA, Single-Stranded