Fig. 4.

Symmetrical cleavage of a cruciform structure by Mus81–Eme1. (a) Schematic of ligation assay. The symmetrical cleavage of a cruciform creates nicked hairpin DNA ends that are substrates for DNA ligase. Linear DNA with sealed hairpins runs as a dimer-length ssDNA circle on a denaturing gel. (b) pAT25 substrate [200 pM] was incubated with either EcME [10 nM] or BamHI (10 units) and followed by heat inactivation. Where indicated, T4 DNA ligase was added to the second incubation. Reaction products were analyzed by either native or denaturing gel electrophoresis and detected by Southern blot. (c) pAT25 substrate [2 nM] was incubated with either wild-type or mutant EcME [30 nM]. The reaction products were used as templates for primer extension reactions by using forward (Fwd) or reverse (Rev) primers as indicated. A Maxam–Gilbert sequencing ladder, run on the same gel, is shown at a lighter exposure. The major EcME cleavage sites on the cruciform in pAT25 are illustrated.