FIGURE 7.

Rolling circle synthesis of leading and lagging strand. A, model of the synthesis of leading and lagging strand synthesis. The annealing of an oligonucleotide containing a 5′ oligo(T)₄₀ tail and an 100-nt stretch (with T, G, and C) to the 200-nt circle (containing A, C, and G) yielded the DNA substrate shown. The addition of T. kodakaraensis PolB, T. kodakaraensis MCM (TK1361), T. kodakaraensis RFC, T. kodakaraensis PCNA, and dNTPs and ATP resulted in the elongation of the leading strand. Following the addition of the T. kodakaraensis DNA primase complex (p41-p46), the reaction was divided into two equal portions. One-half was supplemented with [α-³²P]dTTP, and the other was supplemented with [α-³²P]dATP to monitor the synthesis of leading and lagging strand synthesis as shown in panels B and C. B, leading strand synthesis. Reaction mixtures (20 μl) containing 20 mm Tris-HCl (pH 8.0), 10 mm magnesium acetate, 100 mm NaCl, 1 mm DTT, 100 μg/ml BSA, 5 nm 200-mer DNA circles hybridized to dT₄₀-100-mer, 20 nm T. kodakaraensis RFC, and 50 nm T. kodakaraensis PCNA (TK0535) were prepared on ice. Reactions were supplemented with T. kodakaraensis PolB (44 nm), and the mixture was incubated for 5 min at 60 °C, after which T. kodakaraensis MCM (TK1361) (44 nm), T. kodakaraensis DNA primase complex (74 or 18.5 nm), ATP (2 mm), 150 μm each of dGTP, dCTP, and dATP, and 40 μm [α-³²P]dTTP (18,900 cpm/pmol) was added. The level of T. kodakaraensis DNA primase complex added in lanes 6 and 7 was 74 nm. Reactions were incubated for 10 min at 60 °C, and aliquots were used to measure DNA synthesis and the size of products formed. Alkaline agarose gel (0.6%) electrophoresis was used for the analysis of leading strand synthesis. M, molecular mass markers; inc., incorporation. C, lagging strand synthesis. Reactions were as described in panel A with the exception that 150 μm each of dGTP, dCTP, and dTTP, and 40 μl of [α-³²P]dATP (13,300 cpm/pmol) were added. Alkaline agarose gel (1.1%) electrophoresis was used to analyze the size of the DNA synthesized. The levels of T. kodakaraensis DNA primase added were as described in panel B.