Energy for this unfavorable reaction is provided by the hydrolysis of adenosine triphosphates (ATP) to adenosine diphosphates (ADP) and inorganic phosphate ions (Pi)' In the presence of a single­ stranded DNA binding protein, reannealing of the DNA duplex is prevented.
The helicase depicted here displays a 3 to 5 polarity, tracking unidirectionally along the lower of the two DNA strands in the duplex (the loading strand).

dimers, and multimers (predominantly hexamers) and that they can also be classified based on a substrate requirement for dsDNA, dsRNA, or DNA-RNA hybrids. To unwind dsDNA efficiently, many DNA helicases need to initiate from an ssDNA region adjacent to the duplex part of the substrate molecule. Based on the requirement for an ssDNA overhang of a certain polarity, helicases are divided into two functional groups: those that utilize a 3'-terminated ssDNA are designate as 3' 5' helicases, whereas enzymes that require a 5' overhang are designated as 5' 3' helicases.

Directional Translocation

It is now generally believed that the observed polarity requirement of helicases is a consequence of a directional bias in translocation on ssDNA. For example, the enzyme depicted in Fig. 1 is a 3' 5' helicase. Upon binding to the ssDNA, it starts moving toward the 5' end of the loading strand, which brings the enzyme to the ssDNA-dsDNA junction and subsequently through the duplex portion of the substrate. Evidence for directional translocation on ssDNA was provided by two different approaches. The first examined the dependence of helicase ATPase activity on the length of the ssDNA substrate. The second, based on the ability of many helicases to create sufficient force during ssDNA translocation to disrupt the tight interaction between streptavidin and biotin (Kd= 10-15 M), measured the ability of the helicase to increase the rate of streptavidin dissociation from DNA substrates biotinylated at either the 3' or 5' end. This second method was used successfully to determine the directionality of movement of several helicases on ssDNA.
High-resolution structural data suggest that the helicase signature motifs are not essential for the duplex DNA separation per se, but for the A TP-dependent unidirectional motion of the helicases on either single- or double-stranded DNA lattices. Consequently, it was proposed that the helicase signature motifs define a modular structure that functions as the DNA motor, while additional domains, which may vary from one protein to another, may be responsible for the DNA unwinding.
Accessory factors
Once dsDNA unwinding is achieved, spontaneous reannealing of the duplex may be avoided if the nascent ssDNA strands are trapped by single-stranded DNA binding proteins or other "coupling factors" that hand off the intermediates to the next step in a reaction pathway. Although ssDNA binding proteins have frequently been shown to stimulate helicase activity in vitro, helicase activity can also be stimulated by other accessory factors that increase the rate or processivity of unwinding. The primary replicative helicase of Escherchia coli,DnaB, is a good example of a helicase that acts poorly in isolation from the accessory factors with which the enzyme is intended to operate. As part of the replisome (the DNA synthesis machinery of the cell), the role of DnaB is to separate the DNA strands at the replication fork.
It was shown recently that the rate of movement of the replication machinery at the fork is coordinated by an interaction between DnaB and DNA polymerase (enzyme that synthesizes a daughter strand of DNA residues) that is mediated by the subunit of the DNA polymerase. The subunit bridges the polymerase dimer and the hexameric helicase, inducing a conformational change in DnaB that enhances its translocation rate by almost 30-fold to 1000 base pairs per second. In the absence of, the replication machinery is uncoupled, and the polymerase simply follows DnaB as it unwinds DNA at approximately 35 bp/s. Single-molecule translocation visualization
Until recently, all biochemical data on helicases were derived from conventional bulk-phase techniques, which observe the population-averaged properties of large molecular ensembles. In 2001 two new approaches to visualize translocation by a single molecule of a helicase were reported. These new techniques successfully visualized translocation of a single molecule of RecBCD, a multifunctional heterotrimeric enzyme employed by E. colito initiate homologous recombination at dsDNA breaks. RecBCD is an exceptionally fast helicase that is furnished with all of the processivity and accessory factors it requires. The enzyme has a high affinity for blunt or nearly blunt dsDNA ends, and it can unwind, on average, 30,000 bp

Fig . Tethered particle motion experiment to study DNA translocation by single RecBCD helicase/nuclease molecules. A dsDNA molecule is attached to a glass surface, and RecBCD molecules are attached to, polystyrene beads. As RecBCD tracks along the DNA molecule in an A TP-dependent manner, it gradually draws the bead closer to the glass surface. This translocation results in a decrease in the Brownian motion of the bead that can be measured by light microscopy.

of dsDNA per binding event at a rate of 1000 bp/s, while simultaneously degrading the ssDNA products of its helicase activity.
Tethered particle motion visualization
An alternative single-molecule approach used light microscopy to follow translocation of a biotin tagged RecBCD enzyme bound to a streptavidin-coated polystyrene bead. In the tethered particle motion experiment, dsDNA molecules, modified with digoxigenin at one end, were attached to a glass surface coated with antidigoxigenin antibodies. Bead-labeled RecBCD molecules were bound to the free (unmodified) dsDNA ends. Because the DNA acts as a flexible tether, RecBCD translocation was observed as a decrease in the Brownian motion (the irregular motion of small particles caused by the random bombardment by molecules in the surrounding medium) of the bead as it was pulled toward the glass surface. Combined observations
The two single-molecule experiments are different yet complementary: the tethered particle motion experiment directly measures translocation, whereas the optical trap method (and conventional bulk assays) measures dsDNA unwinding. Therefore, together, the studies provide additional powerful evidence for the coupling of DNA strand separation with movement of the helicase protein on its substrate lattice. Both single-molecule visualization methods show that RecBCD translocates unidirectionally and processively on dsDNA, with each molecule moving at a constant rate (within the limit of experimental detection). Although the average translocation rate is similar to that derived from bulk measurements, considerable variation is observed in the translocation rate of individual RecBCD enzymes. This surprising observation is an example of the kind of information that is accessible only by single-molecule studies.