Fig. 5.

Branched DNA binding and cleavage by 67RuvC R121A and R124A mutant proteins.

A–C. Gel retardation assay showing binding of R121A, R124A and wt 67RuvC to junction (J11, lanes a–d), fork (F11, lanes e–h) and duplex (D11, lanes i–j) DNA substrates. Binding reactions contained 5 mM EDTA, 0.3 nM ³²P-labelled DNA and protein at 2.5, 5 and 10 nM. Samples were incubated on ice for 15 min before separation on 4% PAGE. Lanes a, e and i served as no protein controls.

D–F. DNA branch cleavage assay showing the resolution products of R121A, R124A and wt 67RuvC on junction (J11, lanes a–d), fork (F11, lanes e–h) and duplex (D11, lanes i–j) DNA substrates. Reactions contained 10 mM MgCl₂, 0.3 nM ³²P-labelled DNA and protein at 2.5, 5 and 10 nM. Lanes a, e and i served as no protein controls. Reactions were incubated for 15 min at 37°C before processing and separation on 10% PAGE.