New Insights into Binding of G-segment DNA to the Active Site of Escherichia coli Topoisomerase III.

Escherichia coli topoisomerase III (EcTopo3) is a type IA topoisomerase that binds single-stranded DNA (ssDNA) during DNA cleavage and strand passage. Here, we report five crystal structures of EcTopo3 in complex with distinct 8-base ssDNA oligonucleotides at 1.85-2.22 Å resolution. These structures reveal a previously unrecognized half-open ssDNA-binding mode. In this mode, EcTopo3 engages only the five 3'-terminal nucleotides of the oligonucleotide within the conserved D4/D1 DNA-binding groove, whereas the D1/D3 binding site near the active center remains closed. All five complex structures-three in the open form and two in the half-open form-clearly show that local base binding within the D4/D1 groove is adaptable and involves both direct and water-mediated contacts, consistent with limited sequence specificity. These findings suggest that ssDNA engagement by EcTopo3 may proceed in a stepwise manner, with partial binding in the D4/D1 groove preceding, or occurring independently of, full opening of the D1/D3 site. The half-open structures also identify a distinct metal-binding site on a glycine-rich loop near the active site, occupied by a metal cation coordinated by backbone carbonyls and conserved water molecules. Together, these results reveal greater conformational and mechanistic flexibility in EcTopo3-ssDNA binding than previously appreciated.