Helicases are a class of enzymes that are vital to all organisms. Their main function is to unpack an organism's genetic material. Helicases are motor proteins that move directionally along a nucleic double helix, separating the two hybridized nucleic acid strands (hence helic- + -ase), via the energy gained from ATP hydrolysis. There are many helicases, representing the great variety of processes in which strand separation must be catalyzed. Approximately 1% of eukaryotic genes code for helicases.

The human genome codes for 95 non-redundant helicases: 64 RNA helicases and 31 DNA helicases. Many cellular processes, such as DNA replication, transcription, translation, recombination, DNA repair and ribosome biogenesis involve the separation of nucleic acid strands that necessitates the use of helicases. Some specialized helicases are also involved in sensing viral nucleic acids during infection and fulfill an immunological function. Genetic mutations that affect helicases can have wide-reaching impacts for an organism, due to their significance in many biological processes.

Helicases are often used to separate strands of a DNA double helix or a self-annealed RNA molecule using the energy from ATP hydrolysis, a process characterized by the breaking of hydrogen bonds between annealed nucleotide bases. They also function to remove nucleic acid-associated proteins and catalyze homologous DNA recombination. Metabolic processes of RNA such as translation, transcription, ribosome biogenesis, RNA splicing, RNA transport, RNA editing, and RNA degradation are all facilitated by helicases. Helicases move incrementally along one nucleic acid strand of the duplex with a directionality and processivity specific to each particular enzyme.

Helicases adopt different structures and oligomerization states. Whereas DnaB-like helicases unwind DNA as ring-shaped hexamers, other enzymes have been shown to be active as monomers or dimers. Studies have shown that helicases may act passively, waiting for uncatalyzed unwinding to take place and then translocating between displaced strands, or can play an active role in catalyzing strand separation using the energy generated in ATP hydrolysis. In the latter case, the helicase acts comparably to an active motor, unwinding and translocating along its substrate as a direct result of its ATPase activity. Helicases may process much faster in vivo than in vitro due to the presence of accessory proteins that aid in the destabilization of the fork junction.

Enzymatic helicase action, such as unwinding nucleic acids, is achieved through the lowering of the activation barrier (${\displaystyle B}$) of each specific action. The activation barrier is a result of various factors, and can be defined by

where

Factors that contribute to the height of the activation barrier include: specific nucleic acid sequence of the molecule involved, the number of base pairs involved, tension present on the replication fork, and destabilization forces.

The size of the activation barrier to overcome by the helicase contributes to its classification as an active or passive helicase. In passive helicases, a significant activation barrier exists (defined as ${\displaystyle B>k_{\text{B}}T}$, where ${\displaystyle k_{\text{B}}}$ is the Boltzmann constant and ${\displaystyle T}$ is temperature of the system). Due to this significant activation barrier, its unwinding progression is affected largely by the sequence of nucleic acids within the molecule to unwind, and the presence of destabilization forces acting on the replication fork. Certain nucleic acid combinations will decrease unwinding rates (i.e. guanine and cytosine), while various destabilizing forces can increase the unwinding rate. In passive systems, the rate of unwinding (${\displaystyle V_{un}}$) is less than the rate of translocation (${\displaystyle V_{trans}}$) (translocation along the single-strand nucleic acid, ssNA), due to its reliance on the transient unraveling of the base pairs at the replication fork to determine its rate of unwinding.