(A) Model of a 70 bp bent duplex which spans dimer-related H2TH domains through the TOPRIM/Winged Helix Domain cleavage site of Top6A (using a previously published SAXS model of S. shibatae topo VI with Top6B in an open conformation (Corbett et al., 2007) see also PDB ID: 2ZBK for a similar conformation, stabilized by the inhibitor radicicol (Graille et al., 2008)). Domains are colored as in Figure 3A. DNA was modeled with a continuous bend using web 3DNA (Zheng et al., 2009). (B) Schematic of the estimated duplex lengths needed to span across the H2TH, Stalk/WKxY, and Top6A dimer DNA-binding regions, using the G-segment path modeled in Figure 7A. Note that the Stalk/WKxY region may allow for the asymmetric binding of DNA in different registers, accounting for the jump in affinity seen between 20 and 30 bp DNA duplexes. (C) Nucleotide-dependent cleavage of fluorescein-labeled DNA duplexes by topo VI and mutant constructs. Length-dependent cleavage by wildtype (left), cleavage of a 70 bp duplex by basic-to-neutral mutants (middle), and cleavage by basic-to-acidic mutants (right) was tested. Cleavage reactions containing a 2:1 ratio of enzyme:duplex were run on denaturing PAGE to separate reaction products, and were visualized using a laser gel scanner. Enzyme construct (wildtype, KGRRAAA, KGRREEE, Stalk/WKxYAAA, Stalk/WKxYEEE, H2THAAA, or H2THEEE), duplex length (40 bp, 60 bp, or 70 bp), and addition of 1 mM ATP or 1 mM AMPPNP is noted above each lane. A no enzyme control containing 1 mM AMPPNP and a single strand DNA ladder consisting of 20, 30, 40, 60, 70, and 80 nt oligonucleotides were run for reference. Where present, the percentage of cleavage product relative to intact DNA is quantified above the lane. (D) Nucleotide-dependent bending of a Cy5/Cy5.5-labeled 70 bp duplex was assessed using bulk FRET. Fluorescence emission spectra (left) produced by 630 nm excitation of the Cy5-Cy5.5-labeled DNA show an increase in cy5.5 emission in the presence of topo VI (left, inset) and AMPPNP, but not in the presence of AMPPNP alone. Spectral emission was normalized by total emission from 645 nm to 850 nm. Ratiometric FRET efficiency was monitored over time upon addition of AMPPNP for the noted basic-to-neutral mutant (middle) or basic-to-acidic topo VI mutant (right). Wildtype and duplex alone are shown in each case for comparison. Figure 7—figure supplement 1 confirms FRET changes arise from DNA bending. Figure 7—figure supplement 2 further considers the gate closure activity of KGRRAAA on the 70 bp duplex substrate. Numerical data are reported in Figure 7—source data 1.