Figure 2.

ddGTP halts Okazaki fragment synthesis before completion, but lagging strand synthesis continues. (A) Reactions were carried out under conditions of the optimized rolling circle reaction with ddGTP added at the same time as radiolabeled nucleotide. Okazaki fragments incorporating α-[³²P] dGTP (10 000 cpm/pmol) were monitored by alkaline agarose gel electrophoresis. Average lengths were determined with a cutoff at 20 kb to exclude leading strand products from the quantification. (B) In the presence or absence of 4 µM ddGTP, an optimized rolling circle reaction was conducted as in (A) except that α-[³²P] dCTP (20 000 cpm/pmol) and ddGTP were added at the same time as dNTPs and ATP. The 3 min elongation step before the addition of radioactive nucleotide was skipped so that the products would be short enough for accurate length quantification. Radiolabeled leading strand products were monitored on an alkaline agarose gel. The products of 10, 20 and 30 s reactions were used to calculate the rate of leading strand synthesis. (C) The amount of leading and lagging strand synthesis in the absence or presence of 1 µM ddGTP.