Characterize the interaction of the dna helicase pria with the stalled dna replication fork using atomic force microscopy

Yaqing Wang, Zhiqiang Sun, Piero R. Bianco, Yuri L. Lyubchenko

Research output: Contribution to journalArticlepeer-review

Abstract

In bacteria, the restart of stalled DNA replication forks requires the DNA helicase PriA. PriA can recognize and remodel abandoned DNA replication forks, unwind DNA in the 3'-to-5' direction, and facilitate the loading of the helicase DnaB onto the DNA to restart replication. ssDNA-binding protein (SSB) is typically present at the abandoned forks, protecting the ssDNA from nucleases. Research that is based on the assays for junction dissociation, surface plasmon resonance, single-molecule FRET, and x-ray crystal structure has revealed the helicase activity of PriA, the SSB-PriA interaction, and structural information of PriA helicase. Here, we used Atomic Force Microscopy (AFM) to visualize the interaction between PriA and DNA substrates with or without SSB in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. The protocol describes the steps to obtain high-resolution AFM images and the details of data analysis and presentation.

Original languageEnglish (US)
Article numbere3940
JournalBio-protocol
Volume11
Issue number5
DOIs
StatePublished - Mar 5 2021

Keywords

  • Atomic force microscopy
  • DNA purification
  • DNA replication
  • PriA helicase
  • Protein-DNA interaction
  • SSB protein

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Plant Science

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