Recombinant IntXO protein activity in vitro. (A) Profiles of proteins (5 μl in each lane) synthesized in vitro by use of a PURExpress kit (New England BioLabs). Lane 1, no template DNA; lane 2, DHFR control plasmid used as template DNA; lane 3, pIntCP plasmid used as template DNA. The bottom arrow indicates Escherichia coli dihydrofolate reductase; the top arrow indicates the IntXO protein. Ld, DNA ladder. (B) Determination of IntXO activity in vitro. The agarose gel image shows the pSCP(Ω-km) plasmid treated with proteins synthesized as described for panel A. Lane 1, DNA treated with the proteins from the reaction mixture without template DNA (lane 1 of panel A); lane 2, DNA treated with the proteins from the reaction mixture containing pIntPC template DNA (lane 3 of panel A; see details in Materials and Methods). The diagrams in panel B show the pSCP(Ω-km) substrate resolution, with the IntXO products designated by arrows. Arrowheads on the plasmids indicate the locations and directions of PSL targets.