Figure 4.

RuvAB impose an orientation bias on RuvC-mediated Holliday junction resolution. Synthetic junctions X1 (A) or X2 (B) were 5′-³²P-end labeled in strands 1, 2, 3, or 4, as indicated, and incubated with RuvC (lanes b–e), with RuvB and RuvC (lanes g–j), or with RuvA, RuvB, and RuvC (lanes l–o) for 30 min at 37°C in cleavage buffer, as described in Materials and Methods. RuvA, RuvB, and RuvC were present at 20 nm, 600 nm, and 10 nm, respectively. The DNA concentration was 1 nm. Cleavage products were analyzed by denaturing PAGE, alongside A + G sequence ladders of strand 1 of each junction (lanes a,f,k). The schematic diagram indicates the relative levels of resolution in each orientation.