Figure 1.

The DNA substrate-template construct used for the helicase assay. (A) Outline of the preparative procedure. The flap-containing dsM13 mp18 circle was formed by primer extension under conditions that exclude strand-displacement synthesis, starting with a tailed primer (hybridized to ssM13 mp18 DNA at positions 6288 to 6308) that carries a short single-stranded overhanging DNA sequence [oligo(dT)]. The product was then digested with FspI restriction nuclease, which results in blunt-end cleavage of the DNA at position 6427, to give the 7.25-kb dsDNA molecule carrying a helicase and polymerase assembly site located 120 bp from the end of the fragment. (B) Schematic drawing of the DNA helicase–polymerase substrate-template construct showing details of the sequence at the helicase and polymerase assembly site. Positions are labeled according to the numbering of the M13 mp18 DNA sequence.