Figure 13.6.

Optimizing helicase loading time with annealed oligo. (A) The mean helicase loading time as a function of the length of the 5′ ssDNA tail. Assays measured the loading of 40 nM gp41 at saturating ATP concentration on a 6.8 kbp hairpin substrate. Four oligonucleotides complementary to different sequences along the hairpin were used to obtain 5′ ssDNA tails of approximately 2000, 3000, 4500, and 6000 nt. (B) Schematic representation of the DNA hairpin substrate consisting of a 1239 bp hairpin with a 4-nt loop, a 76-nt 5′-biotinylated ssDNA tail, and a 146-bp 3′-digoxigenin labeled dsDNA tail (Manosas et al., 2009), and the half-hairpin substrate created with a complementary 50-mer oligonucleotide (grey) used to reduce the length of the hairpin and increase the length of the 5′ ssDNA tail.