Atomic force microscopy-based characterization of the interaction of PriA helicase with stalledDNA replication forks

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

Research output: Contribution to journalArticle

Abstract

In bacteria, the restart of stalledDNA replication forks requires theDNA helicase PriA. PriA can recognize and remodel abandonedDNA replication forks, unwindDNA in the 3'-to 5-' direction, and facilitate the loading of the helicase DnaB onto theDNA to restart replication. Single-stranded DNA-binding protein (SSB) is typically present at the abandoned forks, but it is unclear how SSB and PriA interact, although it has been shown that the two proteins interact both physically and functionally. Here, we used atomic force microscopy to visualize the interaction of PriA withDNA substrates with or without SSB. These experiments were done in the absence of ATP to delineate the substrate recognition pattern of PriA before its ATP-catalyzed DNA-unwinding reaction. These analyses revealed that in the absence of SSB, PriA binds preferentially to a fork substrate with a gap in the leading strand. Such a preference has not been observed for 5'-and 3'-tailed duplexes, suggesting that it is the fork structure that plays an essential role in PriA's selection ofDNA substrates. Furthermore, we found that in the absence of SSB, PriA binds exclusively to the fork regions of theDNA substrates. In contrast, fork-bound SSB loads PriA onto the duplexDNA arms of forks, suggesting a remodeling of PriA by SSB. We also demonstrate that the remodeling of PriA requires a functional C-terminal domain of SSB. In summary, our atomic force microscopy analyses reveal key details in the interactions between PriA and stalledDNA replication forks with or without SSB.

Original languageEnglish (US)
Pages (from-to)6043-6052
Number of pages10
JournalJournal of Biological Chemistry
Volume295
Issue number18
DOIs
StatePublished - May 1 2020

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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