The 3 '-tail of a forked-duplex sterically determines whether one or two DNA strands pass through the central channel of a replication-fork helicase

DnaB helicase is a ring-shaped hexamer that unwinds DNA at a replication fo rk. To understand how this protein interacts with DNA during unwinding, Dna B from Thermus aquaticus was incubated with chemically modified forked-dupl ex DNA substrates and the unwinding rates were measured. Unwinding was inhi bited by modifications made to the 5'-tail, but not the 5'-tail, suggesting that the helicase interacts with the 5'-tail but not the 5'-tail during un winding. Using oligonucleotides of mixed polarity, it was confirmed that Dn aB translocates in the 5' to 3' direction as it unwinds DNA. A substrate wa s synthesized that contained two duplexes in tandem. Experiments involving various modifications of this tandem duplex demonstrated that when the 3'-t ail is short, two stands of DNA pass through the central channel of DnaB wi th no resultant unwinding. Thus, the role of the 3'-tail in stimulating unw inding has been elucidated. The 3'-tail does not bind to DnaB during unwind ing, but sterically determines whether one or two DNA strands pass through the central channel of DnaB. Furthermore, a new substrate for DnaB locomoti on has been discovered. DnaB may actively translocate in the 5' to 3' direc tion along single-stranded DNA, even when a complementary strand is also pr esent within the protein's central channel. This new mode of action may reg ulate DnaB activity by inhibiting unwinding at regions of DNA that are not forked. Furthermore, this new function for DnaB may coordinate abortion of leading and lagging strand replication if a nick is encountered on the lead ing strand. (C) 2000 Academic Press.