FIGURE 6.

Duplex DNA ends are not modified by RecN protein. Ligation reactions were carried out in dilute conditions as described under “Experimental Procedures” except that the reaction volume was doubled to 200 μl. 1.2 μm bp of 2.4-kb PstI linearized plasmid DNA (pEAW3) substrate (marker, lds M) was incubated with 2.5 mm ATP in the presence or absence of 2.4 μm Dr RecN protein for 30 min before the addition of 2.5 units of E. coli DNA ligase. Following the ligation step, the E. coli DNA ligase was heat-inactivated (45 min at 65 °C), and each reaction was split into two samples. Both samples were supplemented with PstI restriction endonuclease reaction buffer. The samples were then incubated in the presence (PstI label) or absence (Mock label) of 25 units of PstI endonuclease enzyme for 2 h at 37 °C. The reaction was stopped, and the products were purified as described under “Experimental Procedures.”