Summary information and primary citation
- PDB-id
-
7kik;
DSSR-derived features in text and
JSON formats
- Class
- replication-DNA
- Method
- X-ray (1.73 Å)
- Summary
- Wheat dwarf virus rep domain circular permutation
complexed with a single-stranded DNA 10-mer comprising the
cleavage site and mn2+
- Reference
-
Smiley AT, Tompkins KJ, Pawlak MR, Krueger AJ, Evans 3rd
RL, Shi K, Aihara H, Gordon WR (2023): "Watson-Crick
Base-Pairing Requirements for ssDNA Recognition and
Processing in Replication-Initiating HUH
Endonucleases." Mbio, 14,
e0258722. doi: 10.1128/mbio.02587-22.
- Abstract
- 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 -4 and +1 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
<i>in vitro</i>, we sought to determine to what
extent the canonical -4T·+1A 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.
