Figure 3.

AtPolIs present 3′–5′ exonuclease and 5′–3′ polymerization activities. Exonuclease and polymerization activities measured using a 5′-³²P-labeled 24-mer primer annealed to a complementary 45-mer template DNA. (A) Equimolar amounts of each DNAP were incubated with the labeled primer-template and 3′–5′ exonucleolysis was initiated with the addition of MgCl₂ in the absence of dNTPs. Reactions were stopped at 15, 30, 60, 120 and 240 s. At first-time point (15 s) exonucleolytic degradation of the 24-mer to a 2-mer is observed in the samples incubated with T7DNAP (lane 11), whereas in the samples incubated with AtPolIA and AtPolIB degradation bands corresponding to a 17-mer and a 20-mer are present. The lane labeled with the minus sign (−) corresponds to the reaction without added MgCl₂. (B) Time course reaction from 15 to 240 s showing the polymerase activity of AtPolIA and AtPolIB in comparison to T7DNAP. 5′–3′ polymerization was initiated with the addition of 2 mM MgCl₂ and 100 μM of each dNTP. At the first-time point (15 s), full primer extension to a 45-mer is observed in the samples incubated with T7DNAP and dNTPs (lanes 11–14). In contrast the samples incubated with AtPolIA and AtPolIB present a constant accumulation of the 45-mer from 15 s to 4 min (lanes 1–10).