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

Adam T. Smiley, Kassidy J Tompkins, Matthew R Pawlak, August J. Krueger, Robert L. Evans, Ke Shi, Hideki Aihara, Wendy R. Gordon

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Replication-initiating HUH endonucleases (Reps) are sequence-specific nucleases that cleave and rejoin single-stranded DNA (ssDNA) during rolling-circle replication. These functions are mediated by covalent linkage of the Rep to its substrate post cleavage. Here, we describe the structures of the endonuclease domain from the Muscovy duck circovirus Rep in complex with its cognate ssDNA 10-mer with and without manganese in the active site. Structural and functional analyses demonstrate that divalent cations play both catalytic and structural roles in Reps by polarizing and positioning their substrate. Further structural comparisons highlight the importance of an intramolecular substrate Watson-Crick (WC) base pairing between the 24 and 11 positions. Subsequent kinetic and functional analyses demonstrate a functional dependency on WC base pairing between these positions regardless of the pair's identity (i.e., A T, T A, G C, or C G), highlighting a structural specificity for substrate interaction. Finally, considering how well WC swaps were tolerated in vitro, we sought to determine to what extent the canonical 24T 11A pairing is conserved in circular Rep-encoding single-stranded DNA viruses and found evidence of noncanonical pairings in a minority of these genomes. Altogether, our data suggest that substrate intramolecular WC base pairing is a universal requirement for separation and reunion of ssDNA in Reps.

Original languageEnglish (US)
Issue number1
StatePublished - Jan 2023

Bibliographical note

Funding Information:
W.R.G. received funding from NIH NIGMS (R35 GM119483), A.T.S. received salary support from the Chemistry-Biology Interface Training Grant (T32GM132029), and both K.J.T. and R.L.E. received salary support from the Muscle training grant (T32AR007612). W.R.G. is a Pew Biomedical Scholar; H.A. received funding from NIH (R35 GM118047). This work is based upon research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the National Institutes of Health (P30 GM124165). This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory (DE-AC02-06CH11357).

Publisher Copyright:
© 2022 Smiley et al.


  • covalent adduct
  • HUH endonuclease
  • HUH-seq
  • next-generation sequencing
  • origin of replication
  • protein DNA bioconjugation
  • Rep
  • Watson-Crick base pair

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.


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