Watson-Crick Base-Pairing Requirements for ssDNA Recognition and Processing in Replication-Initiating HUH...

Area of Science:

• Structural Biology
• Virology
• Molecular Biology

Background:

• Replication-initiating HUH endonucleases (Reps) are essential nucleases for rolling-circle replication in circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses.
• These viruses pose significant threats to agriculture and human health, necessitating a deeper understanding of their replication mechanisms.
• Reps mediate DNA cleavage and rejoining through covalent linkage to the substrate, a process targeted for viral inhibition strategies.

Purpose of the Study:

• To elucidate the structural and functional requirements of the Muscovy duck circovirus Rep protein for single-stranded DNA (ssDNA) interaction.
• To investigate the role of divalent cations and intramolecular base pairing in Rep-mediated DNA processing.
• To assess the conservation of these interactions across the CRESS-DNA virus family.

Main Methods:

• Determined crystal structures of the Muscovy duck circovirus Rep endonuclease domain complexed with ssDNA, with and without manganese.
• Performed structural and functional analyses, including kinetic and biochemical assays, to assess the role of cations and base pairing.
• Conducted bioinformatic analysis of CRESS-DNA viral genomes to evaluate the conservation of identified structural features.

Main Results:

• Divalent cations play critical catalytic and structural roles in positioning and polarizing the ssDNA substrate.
• An intramolecular Watson-Crick (WC) base pair between the -4 and +1 positions of the ssDNA is essential for Rep recognition and function, irrespective of the specific base pair identity.
• While noncanonical pairings were observed in a minority of viral genomes, the vast majority of CRESS-DNA origins utilize conserved base pairings at these critical positions.

Conclusions:

• Intramolecular WC base pairing is a universal requirement for ssDNA separation and reunion mediated by Reps in CRESS-DNA viruses.
• Rep-ssDNA interaction exhibits structural specificity, prioritizing base pairing at conserved positions over specific sequence recognition.
• Understanding these conserved structural requirements provides a foundation for developing broad-spectrum antiviral inhibitors targeting CRESS-DNA viruses.