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Cryo-EM structure of AAV2 Rep68 bound to integration site AAVS1: insights into the mechanism of DNA melting

Rahul Jaiswal1, Brandon Braud1, Karen C Hernandez-Ramirez1

  • 1Department of Physiology and Biophysics, Virginia Commonwealth University, School of Medicine, Richmond, VA 23298, United States.

Nucleic Acids Research
|January 30, 2025
PubMed

Insights

Adeno-associated virus Rep68 protein, an SF3 helicase, forms a heptamer for DNA binding. ATP binding induces conformational changes, enabling DNA interactions and distortion for viral replication.

Area of Science:

  • Structural Biology
  • Virology
  • Molecular Biology

Background:

  • The Rep68 protein of Adeno-Associated Virus (AAV) is essential for viral DNA replication.
  • Rep68 functions as a multifunctional SF3 helicase, catalyzing DNA melting and nicking.
  • Understanding Rep68's mechanism is key to AAV lifecycle comprehension.

Purpose of the Study:

  • To elucidate the structural basis of Rep68 assembly and function on DNA.
  • To investigate the role of nucleotide binding in Rep68 conformational changes.
  • To provide insights into the DNA melting process mediated by SF3 helicases.

Main Methods:

  • Cryo-electron microscopy (cryo-EM) was used to determine structures of Rep68.
  • Structures were obtained for Rep68 bound to the AAV integration site 1 in different nucleotide states.
  • Analysis focused on Rep68 complex formation, DNA interactions, and nucleotide-dependent conformational changes.

Main Results:

  • Rep68 forms a heptameric complex around DNA in a nucleotide-free state.
  • Origin-binding domains (OBDs) interact with the Rep-binding element, with additional transient dimers observed.
  • ATPγS binding induces conformational changes, promoting intersubunit and DNA interactions, and DNA distortion via phenylalanine residues.

Conclusions:

  • The heptameric structure is likely crucial for loading Rep68 onto double-stranded DNA.
  • Nucleotide binding triggers significant conformational changes in Rep68, facilitating DNA transactions.
  • These findings offer structural insights into SF3 helicase assembly and DNA melting mechanisms.

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