Fig. 3.

BACH1 preferentially unwinds DNA in the 5′-to-3′ direction. (A) Scheme for helicase directionality assay. A single-stranded 92-mer oligodeoxynucleotide was annealed to M13 DNA and cleaved with SalI, and all available 3′ ends were radiolabeled by using Klenow polymerase to yield a partially double-stranded substrate comprising 38-mer and 55-mer oligonucleotides annealed to linear M13 DNA. (B) Autoradiogram of an 8% polyacrylamide gel of the products generated by incubating the substrate depicted in A with FLAG-BACH1. Lane 1, substrate alone; lane 2, denatured substrate (B); lane 3, 5 ng of UvrD protein as a 3′-to-5′ helicase control; lanes 4–6, substrate plus 60, 150, and 450 ng of FLAG-BACH1.