Helicases are molecular motors that unwind double-stranded DNA or RNA. In addition to unwinding nucleic acids, an important function of these enzymes seems to be the disruption of protein-nucleic acid interactions. Bacteriophage T4 Dda helicase can displace proteins bound to DNA, including streptavidin bound to biotinylated oligonucleotides. We investigated the mechanism of streptavidin displacement by varying the length of the oligonucleotide substrate. We found that a monomeric form of Dda catalyzed streptavidin displacement; however, the activity increased when multiple helicase molecules bound to the biotinylated oligonucleotide. The activity does not result from cooperative binding of Dda to the oligonucleotide. Rather, the increase in activity is a consequence of the directional bias in translocation of individual helicase monomers. Such a bias leads to protein-protein interactions when the lead monomer stalls owing to the presence of the streptavidin block.

The E. coli single-stranded DNA-binding protein (SSB) binds to the fork DNA helicases RecG and PriA in vitro. Typically for binding to occur, 1.3 M ammonium sulfate must be present, bringing into question the validity of these data as these are non-physiological conditions. To determine whether SSB can bind to these helicases, we examined binding in vivo. First, using fluorescence microscopy, we show that SSB localizes PriA and RecG to the vicinity of the inner membrane in the absence of DNA damage. Localization requires that SSB be in excess over the DNA helicases and the SSB C-terminus and both PriA and RecG be present. Second, using purification of tagged complexes, our results demonstrate that SSB binds to PriA and RecG in vivo, in the absence of DNA. We propose that this may be the “storage form” of RecG and PriA. We further propose that when forks stall, RecG and PriA are targeted to the fork by SSB which, by virtue of its high affinity for single stranded DNA, allows these helicases to out compete other proteins. This ensures their actions in the early stages of the rescue of stalled replication forks.

Superfamily 2 helicases are involved in all aspects of RNA metabolism, and many steps in DNA metabolism. This review focuses on the basic mechanistic, structural and biological properties of each of the families of helicases within superfamily 2. There are ten separate families of helicases within superfamily 2, each playing specific roles in nucleic acid metabolism. The mechanisms of action are diverse, as well as the effect on the nucleic acid. Some families translocate on single-stranded nucleic acid and unwind duplexes, some unwind double-stranded nucleic acids without translocation, and some translocate on double-stranded or single-stranded nucleic acids without unwinding.

Background: Pif1 helicase plays a variety of roles in both the nucleus and mitochondria. Results: The kinetic step size for Pif1 is one base pair, and translocation on ssDNA is coupled tightly with ATP hydrolysis. Conclusion: Hydrolysis of one ATP results in movement of Pif1 by a single nucleotide. Significance: Pif1 is an active helicase with a uniform stepping mechanism.

Helicases are molecular motor enzymes that unwind and translocate nucleic acids. One of the central questions regarding helicase activity is whether the process of coupling ATP hydrolysis to DNA unwinding requires an oligomeric form of the enzyme. We have applied a pre-steady-state kinetics approach to address this question with the bacteriophage T4 Dda helicase. If a helicase can function as a monomer, then the burst amplitude in the pre-steady state might be similar to the concentration of enzyme, whereas if the helicase required oligomerization, then the amplitude would be significantly less than the enzyme concentration. DNA unwinding of an oligonucleotide substrate was conducted by using a Kintek rapid quench-flow instrument. The substrate consisted of 12 bp adjacent to 12 nucleotides of single-stranded DNA. Dda (4 nM) was incubated with substrate (16 nM) in buffer, and the unwinding reaction was initiated by the addition of ATP (5 mM) and Mg 2؉ (10 mM). The reaction was stopped by the addition of 400 mM EDTA. Product formation exhibited biphasic kinetics, and the data were fit to the equation for a single exponential followed by a steady state. The amplitude of the first phase was 3.5 ؎ 0.2 nM, consistent with a monomeric helicase. The burst amplitude of product formation was measured over a range of enzyme and substrate concentrations and remained consistent with a functional monomer. Thus, Dda can rapidly unwind oligonucleotide substrates as a monomer, indicating that the functional molecular motor component of a helicase can reside within a single polypeptide.pre-steady-state kinetics M olecular motors are enzymes that transduce chemical energy to mechanical energy. A type of molecular motor that is required for almost all aspects of nucleic acid metabolism is helicase. Helicases are ubiquitous enzymes that unwind or transport double-stranded nucleic acids during replication, recombination, transcription, and DNA repair. The mechanism of this class of enzymes has received a great deal of attention during the past decade (1-4). Helicases are classified in several superfamilies (SFs) based on sequence homology. Because multiple DNA-binding sites are required for processive DNA unwinding, oligomerization can provide a means to provide these sites. Some helicases such as those in the DNA-B-like SF clearly function as hexamers (4). Hexameric helicases can be highly processive, because the DNA passes through a central channel created by the doughnut shape of the enzyme. The fact that many helicases function as hexamers as well as consideration of the necessity for multiple DNA-binding sites has led to the suggestion that oligomerization may be necessary for helicase function. Direct, functional evidence has not been presented for a monomeric helicase that can transduce the chemical energy available from hydrolyzing ATP to mechanical energy for DNA unwinding.