What is Dr. Zubair Khalid's research focus?

Dr. Zubair Khalid specializes in molecular virology, mRNA vaccine development, and computational biology, with a focus on avian pathogens like IBDV and Avian Reovirus.

Where is Dr. Zubair Khalid currently working?

Dr. Zubair Khalid is a Postdoctoral Research Associate at the University of Maryland (UMD), specifically within the Department of Animal and Avian Sciences.

Helicase Activity: Mechanisms, Methods, and Misconceptions

By Dr. Zubair Khalid, DVM, MS, PhD · 2026-08-11

Introduction to Helicase Activity

Helicase activity is the ATP-driven process by which enzymes called helicases separate the two strands of a nucleic acid duplex. This activity is fundamental to virtually every aspect of DNA metabolism, including replication, repair, recombination, and transcription. Without helicases, the double helix would remain stably base-paired, and the genetic information encoded within it would be inaccessible to the polymerases, repair complexes, and regulatory proteins that must read it.

What is a helicase?

A helicase is an enzyme that binds to double-stranded nucleic acids and uses the energy derived from ATP hydrolysis to break the hydrogen bonds between complementary base pairs, thereby separating the strands. The term "helicase" derives from "helix," reflecting the enzyme's role in unwinding the helical structure of DNA or RNA. Helicases are found in all domains of life, from bacteria to humans, and they act on DNA–DNA, RNA–RNA, and DNA–RNA hybrids. For a broader overview of the enzyme class, see the Helicase Definition entry.

The defining biochemical feature of a helicase is its ability to couple nucleotide triphosphate (NTP) hydrolysis to directional movement along a nucleic acid lattice. This movement is called translocation, and it is the mechanical basis for strand separation. Helicases are often described as "molecular motors" because they convert chemical energy into mechanical work, much like kinesin and myosin do in the cytoskeleton.

Role in DNA metabolism

Helicases participate in nearly every process that requires access to single-stranded DNA (ssDNA). During DNA replication, the replicative helicase unwinds the parental duplex ahead of the polymerase, creating the single-stranded templates required for nucleotide incorporation. During DNA repair, helicases unwind damaged regions so that excision nucleases and polymerases can access the lesion. During transcription, RNA polymerase itself contains helicase-like domains that melt the promoter region, and dedicated helicases are required for transcription-coupled repair. In recombination, helicases promote strand exchange by remodeling nucleoprotein filaments.

The importance of helicase activity is underscored by the number of human diseases associated with helicase mutations. Bloom syndrome, Werner syndrome, and Rothmund–Thomson syndrome all result from defects in RecQ-family helicases, and each predisposes patients to cancer. Mutations in the XPD helicase cause xeroderma pigmentosum, a condition characterized by extreme sensitivity to ultraviolet light and a dramatically elevated risk of skin cancer. These clinical associations make helicases not only fascinating enzymes but also important therapeutic targets.

The Molecular Mechanism of Unwinding

The mechanism by which a helicase unwinds DNA can be broken down into three coupled processes: ATP binding and hydrolysis, translocation along the nucleic acid, and strand separation. These processes are tightly linked, and a helicase cannot translocate without hydrolyzing ATP, nor can it unwind without translocating.

ATP hydrolysis and conformational changes

ATP hydrolysis provides the free energy that drives the conformational changes required for movement. A typical helicase cycle proceeds as follows:

ATP binding: The helicase binds ATP in its nucleotide-binding pocket, which is formed by conserved motifs (discussed below). ATP binding induces a conformational change that increases the enzyme's affinity for nucleic acid and positions the motor domains for a power stroke.
Hydrolysis: The γ-phosphate of ATP is cleaved, producing ADP and inorganic phosphate (Pi). This hydrolysis step is catalyzed by a conserved glutamate residue that activates a water molecule for nucleophilic attack on the γ-phosphate.
Product release: The release of Pi, followed by ADP, triggers a second conformational change that resets the enzyme to its low-affinity state, allowing it to release the nucleic acid and begin a new cycle.

The key point is that ATP binding and hydrolysis are coupled to changes in the helicase's affinity for ssDNA versus double-stranded DNA (dsDNA), as well as to changes in the relative positions of its domains. This coupling is often described by a "Brownian ratchet" or "power stroke" model. In the power stroke model, ATP binding drives a large conformational change that physically pushes the helicase forward. In the Brownian ratchet model, thermal fluctuations allow the helicase to move forward and backward, but ATP hydrolysis biases the equilibrium toward forward movement by preventing backward sliding.

Translocation along DNA

After ATP hydrolysis, the helicase must move along the nucleic acid. Translocation is directional and occurs in a stepwise manner, with each ATP hydrolyzed corresponding to the movement of one or two nucleotides. The step size varies among helicases: the replicative helicase DnaB from E. coli translocates with a step size of approximately one nucleotide per ATP, while the eukaryotic Mcm2-7 complex moves in steps of two nucleotides.

Translocation requires that the helicase maintain contact with the nucleic acid at all times. This is achieved through a "hand-over-hand" mechanism in which two or more domains alternately bind and release the DNA. At any given moment, at least one domain is tightly bound to the nucleic acid, preventing the helicase from diffusing away. This alternating binding is analogous to the way a person climbs a rope: one hand grips while the other reaches forward.

Strand separation and processivity

Strand separation occurs at the leading edge of the helicase, where the enzyme contacts the duplex junction. The mechanism of separation depends on the helicase's architecture. Some helicases, such as DnaB and the Mcm2-7 complex, form ring-shaped hexamers that encircle one strand of the DNA. These "ring helicases" translocate along one strand and sterically exclude the complementary strand, effectively peeling it away from the duplex. Other helicases, such as RecQ and UvrD, are monomeric or dimeric and use a "wedge" or "plowshare" mechanism, in which a protein domain physically separates the two strands as the enzyme moves along the DNA.

Processivity is the number of base pairs a helicase can unwinding before dissociating from its substrate. This parameter varies widely. The replicative helicase DnaB is highly processive, unwinding tens of thousands of base pairs in a single binding event. In contrast, repair helicases such as UvrD are poorly processive, unwinding only 20–50 base pairs at a time. Processivity is enhanced by accessory proteins, such as single-stranded DNA binding proteins (SSBs), which coat the newly exposed ssDNA and prevent it from reannealing. In E. coli, the SSB protein stimulates DnaB processivity by preventing the formation of secondary structures in the unwound ssDNA. For more detail on the mechanics of strand separation, see Helicase Break Hydrogen Bonds.

Structural Features of Helicases

Despite their functional diversity, all helicases share a set of conserved structural motifs that are responsible for ATP binding and hydrolysis. These motifs were first identified by sequence alignment and later confirmed by X-ray crystallography and cryo-electron microscopy.

Helicase superfamilies

Helicases are classified into six superfamilies (SF1–SF6) based on the presence and arrangement of conserved sequence motifs. The two largest and best-characterized superfamilies are SF1 and SF2, which contain non-ring helicases that function as monomers or dimers. SF3–SF6 contain ring-forming helicases, including the replicative helicases of bacteria (SF4), archaea, and eukaryotes (SF3 and SF6).

All helicases contain at least the following conserved motifs:

Walker A motif (P-loop): A conserved sequence of the form GxxxxGKT/S, where x is any amino acid. This motif binds the phosphate groups of ATP and is essential for nucleotide binding.
Walker B motif: A conserved sequence of the form hhhhDExx, where h is a hydrophobic residue. The aspartate coordinates a magnesium ion (Mg²⁺) required for catalysis, and the glutamate activates the water molecule that attacks the γ-phosphate.
Arginine finger: A conserved arginine residue that senses the presence of the γ-phosphate and transmits the nucleotide state to the rest of the protein.

In addition to these core motifs, SF1 and SF2 helicases contain a set of auxiliary motifs (Ia, Ib, II, III, IV, V, VI) that are involved in nucleic acid binding and in coupling ATP hydrolysis to conformational changes. The presence and arrangement of these motifs determine whether a helicase belongs to SF1 or SF2. SF1 helicases, such as Rep and UvrD, translocate with a 3′ to 5′ polarity, while SF2 helicases, such as RecQ and NS3, can have either polarity.

Oligomeric state and active site architecture

Helicases function as monomers, dimers, or hexamers, and their oligomeric state is intimately linked to their mechanism. Monomeric helicases, such as the SF1 helicase PcrA, can translocate along ssDNA but are poorly processive. Dimeric helicases, such as Rep, use a "rolling" mechanism in which the two subunits alternately bind and release the DNA, allowing the enzyme to move along the duplex without fully dissociating.

Hexameric helicases, which include the replicative helicases, form ring-shaped structures with a central channel through which one strand of DNA passes. The channel is lined with loops that contact the sugar-phosphate backbone of the DNA, and ATP hydrolysis drives the sequential movement of these loops, propelling the DNA through the channel. The hexameric architecture provides high processivity because the DNA is topologically trapped within the ring, making dissociation unlikely. For a detailed description of the structural features, see the Helicase Structure resource.

The active site of a hexameric helicase is formed at the interface between adjacent subunits. Each subunit contributes a Walker A motif from one side and an arginine finger from the neighboring subunit. This arrangement means that ATP hydrolysis is coordinated across the ring, with subunits hydrolyzing ATP in a sequential or stochastic manner. In the Mcm2-7 complex, the six subunits are non-identical, and only a subset of the active sites are catalytically competent. This asymmetry is thought to allow for regulation of helicase activity during the cell cycle.

Polarity and Directionality

Helicases are directional enzymes: they translocate along a nucleic acid strand in a defined orientation. This directionality is referred to as polarity and is a fundamental property that determines the biological function of a helicase.

Defining polarity

Polarity is defined by the direction of translocation relative to the sugar-phosphate backbone of the nucleic acid. A helicase that moves along a single strand in the 3′ to 5′ direction is said to have 3′→5′ polarity, while one that moves in the 5′ to 3′ direction has 5′→3′ polarity. It is important to note that polarity is defined with respect to the strand on which the helicase translocates, not the strand that is displaced.

At a replication fork, the leading strand is synthesized continuously in the 5′ to 3′ direction, while the lagging strand is synthesized discontinuously in short Okazaki fragments. The replicative helicase must unwind the duplex ahead of the polymerase, and its polarity determines which strand it encircles. In E. coli, DnaB has 5′→3′ polarity and encircles the lagging strand. In eukaryotes, the Mcm2-7 complex also has 3′→5′ polarity and encircles the leading strand. The choice of strand is not arbitrary: it positions the helicase so that it unwinds the duplex in the correct direction relative to the polymerase.

Methods to determine directionality

The polarity of a helicase can be determined experimentally using a variety of assays. The most common approach is a strand displacement assay with a substrate that has a defined polarity. For example, a partial duplex substrate can be constructed with a short oligonucleotide annealed to a longer template, leaving a single-stranded tail of known polarity. If the helicase translocates 3′→5′, it will load onto a 3′ tail and displace the duplex; if it translocates 5′→3′, it will load onto a 5′ tail.

A more sophisticated method uses a "helicase roadblock" assay, in which a streptavidin–biotin complex is placed on the single-stranded tail to block translocation. By placing the roadblock at different positions, one can determine the direction of movement. Single-molecule approaches, such as optical tweezers or magnetic tweezers, can also be used to directly observe the direction of translocation in real time. These methods have revealed that some helicases can switch polarity under certain conditions, although this is rare and usually requires specific cofactors.

Helicases in DNA Replication and Repair

Helicases are central players in DNA replication and repair, and specific helicases have evolved to perform specialized functions in these pathways.

Replicative helicases

The replicative helicase is the enzyme that unwinds the parental duplex at the replication fork. In bacteria, the replicative helicase is DnaB, a hexameric ring with 5′→3′ polarity. DnaB is loaded onto the DNA at the origin of replication by the DnaC loader protein, which uses ATP to open the ring and place it around the lagging strand. Once loaded, DnaB translocates along the lagging strand, unwinding the duplex and providing ssDNA templates for the leading and lagging strand polymerases. The rate of DnaB translocation is approximately 500–1000 base pairs per second, which is well matched to the rate of fork progression.

In eukaryotes, the replicative helicase is the Mcm2-7 complex, a heterohexamer of six related subunits (Mcm2 through Mcm7). Unlike DnaB, Mcm2-7 has 3′→5′ polarity and encircles the leading strand. Mcm2-7 is loaded onto the DNA during G1 phase as an inactive double hexamer, and it is activated only at the G1/S transition by the action of the kinases Cdc7 and CDK2. Activation requires the recruitment of additional factors, including Cdc45 and the GINS complex, which together form the CMG complex (Cdc45, Mcm2-7, GINS). The CMG complex is the active replicative helicase in eukaryotic cells, and it unwinds DNA at a rate of approximately 20–50 base pairs per second. For more on the replicative helicase, see Replication Fork Helicase.

Repair helicases and their associated diseases

In addition to their roles in replication, helicases are essential for DNA repair. The RecQ family of helicases is particularly important in maintaining genome stability. Humans have five RecQ helicases: RECQ1, BLM, WRN, RECQ4, and RECQ5. Mutations in BLM cause Bloom syndrome, characterized by growth retardation, immunodeficiency, and a predisposition to many types of cancer. BLM helicase, together with topoisomerase IIIα and the structural proteins RMI1 and RMI2, forms the BTR complex, which dissolves double Holliday junctions during homologous recombination. This dissolution activity prevents crossing over and reduces the risk of loss of heterozygosity, which is why BLM mutations lead to cancer predisposition.

WRN helicase, which is mutated in Werner syndrome, is unusual in that it possesses both helicase and exonuclease activities. Werner syndrome is characterized by premature aging, and cells from affected individuals show genomic instability and accelerated telomere shortening. WRN helicase is thought to function in the repair of replication-associated double-strand breaks and in the resolution of aberrant replication intermediates.

The XPD helicase is a component of the transcription factor TFIIH, which is required for both transcription and nucleotide excision repair (NER). XPD has 5′→3′ helicase activity and unwinds DNA around a lesion during NER. Mutations in XPD cause xeroderma pigmentosum, trichothiodystrophy, and Cockayne syndrome, depending on the specific mutation and its effect on protein stability and activity.

Experimental Methods to Study Helicase Activity

Studying helicase activity requires assays that can detect strand separation, ATP hydrolysis, or translocation. Each method has its strengths and limitations, and the choice of assay depends on the specific question being asked.

In vitro unwinding assays

The most direct assay for helicase activity is the gel-based unwinding assay. In this assay, a radiolabeled or fluorescently labeled oligonucleotide is annealed to a complementary template to form a partial duplex. The helicase is added in the presence of ATP and a protein trap (usually an excess of unlabeled ssDNA) to prevent reannealing of the displaced strand. After incubation at 37°C for 10–30 minutes, the reaction is stopped by adding SDS and EDTA, and the products are separated by native polyacrylamide gel electrophoresis. The displaced single-stranded oligonucleotide migrates faster than the duplex, allowing quantification of the unwinding reaction.

A typical unwinding reaction contains 1–10 nM helicase, 0.5–1 nM DNA substrate, 1–5 mM ATP, and a buffer containing 20 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl₂, and 1 mM DTT. The reaction is initiated by adding ATP and MgCl₂, and the extent of unwinding is quantified by phosphorimaging or fluorescence imaging.

ATPase assays measure the rate of ATP hydrolysis, which is coupled to translocation and unwinding. The most common ATPase assay uses [γ-³²P]ATP and thin-layer chromatography to separate the radiolabeled Pi from the unhydrolyzed ATP. Alternatively, a colorimetric assay using malachite green can detect the release of inorganic phosphate. ATPase assays are useful for determining the kinetic parameters of a helicase, such as the Michaelis–Menten constant (Km) for ATP and the turnover number (kcat).

Single-molecule approaches

Single-molecule techniques have revolutionized the study of helicases by allowing direct observation of individual enzyme molecules. Optical tweezers use a focused laser beam to trap a bead attached to one end of a DNA molecule. As the helicase unwinds the DNA, the change in DNA length is measured by the movement of the bead, providing a real-time readout of unwinding. This technique can measure the step size, velocity, and processivity of a helicase with nanometer and millisecond resolution.

Fluorescence resonance energy transfer (FRET) is another powerful single-molecule approach. In a FRET-based unwinding assay, a donor fluorophore is attached to one strand of the duplex and an acceptor fluorophore to the other. When the duplex is intact, the fluorophores are close together, and FRET is efficient. As the helicase unwinds the DNA, the fluorophores separate, and FRET decreases. This decrease can be monitored in real time, allowing the observation of individual unwinding events. For a practical overview of these methods, see the Helicase Enzyme page.

Regulation of Helicase Activity

Helicase activity must be tightly regulated to ensure that DNA unwinding occurs at the right time and place. Dysregulation of helicase activity can lead to genomic instability, replication stress, and cancer.

Accessory factors and loaders

Many helicases require accessory proteins for loading onto the DNA and for activation. The bacterial replicative helicase DnaB is loaded onto the origin by DnaC, which binds to the DnaB hexamer and delivers it to the DnaA–origin complex. DnaC uses ATP to open the DnaB ring, and after loading, ATP hydrolysis by DnaC triggers its dissociation from DnaB. In eukaryotes, the Mcm2-7 complex is loaded onto the origin by the origin recognition complex (ORC), Cdc6, and Cdt1. This loading occurs only during G1 phase, when CDK activity is low, ensuring that replication origins are licensed only once per cell cycle.

Accessory factors also regulate helicase processivity. The CMG complex, which is the active form of the eukaryotic replicative helicase, contains the accessory factors Cdc45 and GINS, which stabilize the Mcm2-7 ring and increase its processivity. In bacteria, the τ subunit of DNA polymerase III holoenzyme interacts with DnaB and stimulates its helicase activity, coupling unwinding to DNA synthesis.

Phosphorylation and other modifications

Post-translational modifications play a critical role in regulating helicase activity. The Mcm2-7 complex is phosphorylated by the kinases Cdc7 and CDK2 at the G1/S transition, and this phosphorylation is required for helicase activation. Phosphorylation of Mcm2-7 promotes the recruitment of Cdc45 and GINS, leading to the formation of the active CMG complex. In contrast, phosphorylation of Mcm2-7 by CDK2 during S phase prevents re-loading of the helicase at origins that have already fired, ensuring that DNA is replicated only once per cell cycle.

Ubiquitination also regulates helicase function. The BLM helicase is ubiquitinated and degraded in response to DNA damage, and this degradation is thought to prevent inappropriate recombination. Conversely, deubiquitination of BLM by USP10 stabilizes the protein and promotes its function in homologous recombination.

Common Pitfalls and Misconceptions

Students frequently encounter several conceptual difficulties when studying helicases. Understanding these pitfalls is essential for mastering the material.

Helicase vs. topoisomerase

A common error is to confuse helicases with topoisomerases. Both enzymes act on DNA, but they solve different problems. Helicases break the hydrogen bonds between base pairs, separating the two strands of the duplex. Topoisomerases, in contrast, break and rejoin the phosphodiester backbone of one or both DNA strands to relieve torsional stress. Topoisomerases do not separate the strands; they change the linking number of the DNA. The two enzymes often work together: helicases create positive supercoils ahead of the replication fork, and topoisomerases relieve this superhelical tension. For a direct comparison, see Helicase a Topoisomerase.

ATP hydrolysis is not always coupled to unwinding

Another misconception is that ATP hydrolysis is always directly coupled to strand separation. While ATP hydrolysis is required for translocation, some helicases can bind and melt a duplex without hydrolyzing ATP. For example, the DEAD-box RNA helicases can unwind short RNA duplexes using only ATP binding, not hydrolysis. In these cases, ATP binding induces a conformational change that destabilizes the duplex, and ATP hydrolysis is required for enzyme recycling rather than for the unwinding step itself. This is an important distinction, as it means that an ATPase-deficient helicase can still unwind a substrate in some contexts.

All helicases are not processive

Students often assume that all helicases are highly processive, unwinding thousands of base pairs in a single binding event. In reality, processivity varies widely among helicases. Repair helicases such as UvrD and Rep are poorly processive, unwinding only tens of base pairs before dissociating. This low processivity is functionally important: repair helicases need to unwind only a short region around a lesion, and their repeated cycles of binding, unwinding, and dissociation allow them to survey the DNA for damage. In contrast, replicative helicases are highly processive, and their processivity is enhanced by accessory factors.

Summary and Study Tips

Helicase activity is a central topic in molecular biology, and a solid understanding of the mechanisms, methods, and regulation of helicases is essential for any student of the field.

Key takeaways

Helicases are ATP-driven molecular motors that unwind nucleic acid duplexes.
ATP binding and hydrolysis drive conformational changes that result in directional translocation along the nucleic acid.
Helicases are classified into superfamilies based on conserved sequence motifs, and their oligomeric state (monomer, dimer, hexamer) determines their mechanism and processivity.
Polarity (3′→5′ or 5′→3′) is a fundamental property that determines the biological function of a helicase.
Replicative helicases (DnaB, Mcm2-7) are highly processive and are regulated by accessory proteins and post-translational modifications.
Helicase activity is measured using unwinding assays, ATPase assays, and single-molecule techniques.
Helicases are distinct from topoisomerases, and ATP hydrolysis is not always directly coupled to unwinding.

Exam-focused review questions

Describe the ATP hydrolysis cycle of a helicase and explain how it is coupled to translocation.
Compare and contrast the mechanisms of ring-shaped hexameric helicases and monomeric helicases.
What is the difference between 3′→5′ and 5′→3′ polarity, and how is polarity determined experimentally?
Explain the role of the Mcm2-7 complex in eukaryotic DNA replication and how it is regulated.
What are the clinical consequences of mutations in RecQ helicases?
Design an experiment to determine whether a newly discovered helicase has 3′→5′ or 5′→3′ polarity.
Why is it incorrect to say that helicases and topoisomerases perform the same function?

Frequently Asked Questions

What is helicase activity?

Helicase activity is the ATP-dependent unwinding of double-stranded nucleic acids into single strands. It is catalyzed by helicase enzymes, which translocate along one strand of the duplex and disrupt the hydrogen bonds between base pairs. This activity is essential for DNA replication, repair, recombination, and transcription.

How does a helicase unwind DNA?

A helicase unwinds DNA by coupling ATP hydrolysis to conformational changes that drive directional movement along the nucleic acid. The helicase binds to a single-stranded region of the DNA, hydrolyzes ATP, and uses the energy to translocate along the strand. As it moves, it physically separates the two strands of the duplex. Ring-shaped helicases encircle one strand and exclude the other, while non-ring helicases use a wedge domain to split the duplex.

What is the directionality of helicase activity?

Directionality, or polarity, refers to the direction of translocation along the sugar-phosphate backbone. A helicase with 3′→5′ polarity moves along a single strand from the 3′ end toward the 5′ end, while a helicase with 5′→3′ polarity moves in the opposite direction. Polarity is determined experimentally using partial duplex substrates with single-stranded tails of defined polarity.

What is the role of ATP in helicase activity?

ATP provides the chemical energy that drives the conformational changes required for translocation and strand separation. ATP binding induces a conformational change that increases the helicase's affinity for nucleic acid, and ATP hydrolysis triggers a second conformational change that resets the enzyme. The free energy of ATP hydrolysis is used to bias the helicase's movement in a forward direction.

What are the main types of helicases?

Helicases are classified into six superfamilies (SF1–SF6) based on conserved sequence motifs and structural features. SF1 and SF2 contain non-ring helicases that function as monomers or dimers, while SF3–SF6 contain ring-forming hexameric helicases. Examples include DnaB (bacterial replicative helicase), Mcm2-7 (eukaryotic replicative helicase), RecQ (repair helicase), and NS3 (viral RNA helicase).

How is helicase activity measured in the lab?

Helicase activity is measured using several complementary assays. Gel-based unwinding assays detect the displacement of a labeled oligonucleotide from a partial duplex. ATPase assays measure the rate of ATP hydrolysis using radiolabeled ATP or colorimetric detection of inorganic phosphate. Single-molecule techniques, such as optical tweezers and FRET, allow direct observation of unwinding in real time.

What is the difference between helicase and topoisomerase?

Helicases and topoisomerases both act on DNA but perform distinct functions. Helicases break hydrogen bonds between base pairs to separate the two strands of the duplex. Topoisomerases break and rejoin the phosphodiester backbone to change the linking number of the DNA, relieving torsional stress. Helicases create topological problems that topoisomerases resolve, and the two enzymes often work together during DNA replication.

Key Takeaways

Helicase activity is the ATP-driven unwinding of nucleic acid duplexes, essential for replication, repair, and transcription.
ATP binding and hydrolysis drive conformational changes that produce directional translocation along the nucleic acid.
Helicases are classified into superfamilies (SF1–SF6) based on conserved motifs, and their oligomeric state determines their mechanism and processivity.
Polarity (3′→5′ or 5′→3′) is a fundamental property that determines the biological role of a helicase.
Replicative helicases (DnaB, Mcm2-7) are highly processive and tightly regulated by accessory factors and post-translational modifications.
Helicase activity is measured using unwinding assays, ATPase assays, and single-molecule techniques such as optical tweezers and FRET.
Helicases are distinct from topoisomerases, and ATP hydrolysis is not always directly coupled to strand separation.