Figure 1.

(A) The structure and nucleotide sequence of the minicircle primer-template is shown. The minicircle was constructed as described in Materials and Methods. It consists of a 70-base minicircle to which a 109-base lagging-strand template is annealed to provide a 40-base 5′-single-stranded tail to which the gp41 helicase can bind. Two gp61 primase recognition sites (5′-GTT-3′), shown in bold) are included for the synthesis of the pentaribonucleotide primers required to initiate Okazaki fragment synthesis. The sequence of the minicircle (leading-strand template) is devoid of guanine residues, and the lagging-strand template (109-nt oligonucleotide) is devoid of cytosine residues, allowing leading- and lagging-strand synthesis to be independently quantitated by measuring the incorporation of dGMP and dCMP, respectively. (B) DNA synthesis on the minicircle template was performed and analyzed by alkaline gel electrophoresis as described in Materials and Methods. The reaction contained either [α-³²P]dGTP or [α-³²P]dCTP, as indicated, to follow the products of leading- and lagging-strand synthesis, respectively.