Hairpin Construct Synthesis for Helicase Studies

The hairpin construct was synthesized^{28 (link),35 (link)} by ligating a variable 89-bp hairpin stem capped by a (dT)₄ tetraloop to two 1.5-kb double-stranded handles made by PCR amplification of sections of the pBR322 plasmid (New England Biolabs, Ipswitch, MA, USA). The left and right handles were respectively modified with 5′ biotin and digoxigenin to facilitate attachment to streptavidin- and anti-digoxigenin antibody-coated beads. In the final construct, the hairpin insert was flanked on the 3′ side by a poly-dT loading site for helicase binding (Fig. 1b). For experiments probing the stepping behavior of monomeric UvrD, the loading site was 10 nt long to ensure that only a monomer of UvrD bound to the DNA. A longer loading site of 19, 38, or 60 nt was used for experiments examining the stepping dynamics of dimeric UvrD. All oligonucleotides were purchased from Integrated DNA Technologies (Coralville, IA, USA). Sequences for all hairpin inserts and primers are displayed in Supplementary Table 1.