Figure 5.

Exonuclease activity of ttRecJ and cd-ttRecJ. (A) An aliquot of 50 nM 5′-³²P-labeled ssDNA was reacted with 0.1 µM ttRecJ at 37°C. The reaction mixture contained 20 mM Tris–HCl, 10 mM MgCl₂, 100 mM KCl and 1 mM DTT, pH 7.5. The degradation of ssDNA was analyzed by denaturing PAGE (15% polyacrylamide). Lane 1, ssDNA; lanes 2–6, ssDNA reacted with ttRecJ for the incubation period indicated above each lane. The 49nt arrow indicates the substrate 49mer ssDNA and the 1 nt arrow the released 5′-end nucleotide. (B) An aliquot of 20 nM 5′-³²P-labeled ssDNA was reacted with 0.1 µM ttRecJ and cd-ttRecJ at 25°C for 30 min. The reaction mixture contained 20 mM Tris–HCl and 100 mM KCl, pH 7.5, with the addition of 10 mM MgC1₂ or MnCl₂. Lanes 1 and 5, ssDNA; lane 2, with ttRecJ; lane 3, with ttRecJ in the presence of MgC1₂; lane 4, with ttRecJ in the presence of MnCl₂; lane 6, with cd-ttRecJ; lane 7, with cd-ttRecJ in the presence of MgC1₂; lane 8, with cd-ttRecJ in the presence of MnCl₂. (C) Time course of ttRecJ exonuclease activity. The reaction mixture was as in (A). The percentage of undegraded DNA was plotted against incubation time. Open circle, 37°C; closed circle, 50°C.