A HEXAMERIC HELICASE ENCIRCLES ONE DNA STRAND AND EXCLUDES THE OTHER DURING DNA UNWINDING

The bacteriophage T7 DNA helicase/primase (gene 4 protein) is a ring-l ike hexamer that encircles ssDNA and requires forked DNA to catalyze D NA unwinding. we report that optimal rates of unwinding of forked DNA require ssDNA tails of 55 nucleotides on the 5'-to-3' strand and 15 nu cleotides on the 3'-to-5' strand, Surprisingly, streptavidin bound to a biotinylated 3'-end fully substitutes for the 3'-to-5' ssDNA tail, T his suggests that excluding the 3'-to-5' DNA strand from the center of the helicase is an essential aspect of the mechanism of hexameric hel icase-catalyzed DNA unwinding. We also report that streptavidin bound to a biotinylated dT within the 5'-to-3' strand of the duplexed region abolishes DNA unwinding; whereas, streptavidin bound to a biotinylate d dT within the duplexed region of the other strand has no effect. The se results unambiguously demonstrate that the T7 gene 4 protein is a 5 '-to-3' helicase and imply that during DNA unwinding the 5'-to-3' stra nd transverses the center of the ring while the 3'-to-5' strand is exc luded from the center of the ring. Implications for collisions between a helicase and other protein-DNA complexes are discussed.