DnaB from Thermus aquaticus unwinds forked duplex DNA with an asymmetric tail length dependence

DnaB helicase is a ring-shaped hexamer of 300 kDa that is essential for rep lication of the bacterial chromo some. The dnaB gene from Thermus aquaticus was isolated and cloned, and its gene product was expressed and purified t o homogeneity, A helicase assay was developed, and optimal conditions for T , aquaticus DnaB activity were determined using a forked duplex DNA substra te. The activity required a hydrolyzable nucleoside triphosphate and both 5 '- and 3'-single-stranded DNA tail regions, Under conditions of single enzy matic turnover, the lengths of the 5'- and S'-single-stranded regions were varied, and 6-10 nucleotides of the 5'-single-stranded tail and 21-30 nucle otides of the 3'-single-stranded tail markedly stimulated the unwinding rat e. These data suggest that DnaB from T, aquaticus interacts with both DNA s ingle-stranded tails during unwinding and that a greater portion of the 3'- tail is in contact with the protein. Two models are consistent with these d ata. In one model, the 5'-single stranded region passes through the central hole of the DnaB ring, and the 3'-tail makes extensive contact with the ou tside of the protein. In the other model, the 3'-single-stranded region pas ses through the DnaB ring, and the outside of the protein contacts the 5'-t ail.