An assay for determining the direction of DNA helicase activity. (a) DnaB helicase is added to a long, single-stranded DNA that has short ³²P-labeled DNA strands of different sizes annealed to its ends (shown here are a 796-mer and 722-mer). Each annealed DNA strand has a short single-stranded tail to mimic a replication fork. DnaB initially binds to the single-stranded DNA region, then translocates in one direction to unwind one of the two annealed-DNA duplexes. (b) The DNA-unwinding products are analyzed in a polyacrylamide gel. The result here shows that DnaB displaced only the 722-mer, revealing that DnaB translocates in the 5′→3′ direction along single-stranded DNA.