FIGURE 4.

The TREX1 3′ exonuclease has dsDNA degradation activity. The supercoiled dsDNA plasmids 1 (A, lane 2) and 2 (B, lane 8) were nicked by incubating with Nt.BbvCI restriction enzyme (A, lane 3; and B, lane 9) and purified as described under “Experimental Procedures.” The plasmid 2 was linearized by incubating with EcoRI (C, lane 2) or SacI (D, lane 8) and purified. Exonuclease time course reactions (100 μl) were prepared containing nicked dsDNA plasmid 1 (A) or 2 (B) or linearized dsDNA plasmid 2 (C and D) and 7.6 nm TREX1^WT. The samples (20 μl) were removed after incubation for the indicated times (A, lanes 4–7; and B, lanes 10–13). The reaction products were subjected to electrophoresis on agarose gels, and the amounts of dNMP excised by TREX1^WT were estimated as described under “Experimental Procedures.” The positions of migration of Form I supercoiled dsDNA (dsDNA), Form II nicked dsDNA (Nicked dsDNA), Form III linear dsDNA (linear dsDNA), and circular ssDNA (ssDNA) are indicated. Lane 1 contains the 1-kb ladder (Invitrogen).