Helicase


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, 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.

Function

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.

Activation barrier in helicase activity

Enzymatic helicase action, such as unwinding nucleic acids, is achieved through the lowering of the activation barrier of each specific action. The activation barrier is a result of various factors, and can be defined by
where
  • = number of unwound base pairs,
  • = free energy of base pair formation,
  • = reduction of free energy due to helicase, and
  • = reduction of free energy due to unzipping forces.
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.

Active and passive helicases

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. 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, while various destabilizing forces can increase the unwinding rate. In passive systems, the rate of unwinding is less than the rate of translocation , due to its reliance on the transient unraveling of the base pairs at the replication fork to determine its rate of unwinding.
In active helicases,, where the system lacks a significant barrier, as the helicase can destabilize the nucleic acids, unwinding the double-helix at a constant rate, regardless of the nucleic acid sequence. In active helicases, is closer to, due to the active helicase ability to directly destabilize the replication fork to promote unwinding.
Active helicases show similar behaviour when acting on both double-strand nucleic acids, dsNA, or ssNA, in regards to the rates of unwinding and rates of translocation, where in both systems and are approximately equal.
These two categories of helicases may also be modeled as mechanisms. In such models, the passive helicases are conceptualized as Brownian ratchets, driven by thermal fluctuations and subsequent anisotropic gradients across the DNA lattice. The active helicases, in contrast, are conceptualized as stepping motors – also known as powerstroke motors – utilizing either a conformational "inch worm" or a hand-over-hand "walking" mechanism to progress. Depending upon the organism, such helix-traversing progress can occur at rotational speeds in the range of 5,000 to 10,000 R.P.M.

History of DNA helicases

DNA helicases were discovered in E. coli in 1976. This helicase was described as a "DNA unwinding enzyme" that is "found to denature DNA duplexes in an ATP-dependent reaction, without detectably degrading". The first eukaryotic DNA helicase discovered was in 1978 in the lily plant. Since then, DNA helicases were discovered and isolated in other bacteria, viruses, yeast, flies, and higher eukaryotes. To date, at least 14 different helicases have been isolated from single celled organisms, 6 helicases from bacteriophages, 12 from viruses, 15 from yeast, 8 from plants, 11 from calf thymus, and approximately 25 helicases from human cells. Below is a history of helicase discovery:
  • 1976 – Discovery and isolation of E. coli-based DNA helicase
  • 1978 – Discovery of the first eukaryotic DNA helicases, isolated from the lily plant
  • 1982 – "T4 gene 41 protein" is the first reported bacteriophage DNA helicase
  • 1985 – First mammalian DNA helicases isolated from calf thymus
  • 1986 – SV40 large tumor antigen reported as a viral helicase
  • 1986 – ATPaseIII, a yeast protein, determined to be a DNA helicase
  • 1988 – Discovery of seven conserved amino acid domains determined to be helicase motifs
  • 1989 – Designation of DNA helicase Superfamily I and Superfamily II
  • 1989 – Identification of the DEAD box helicase family
  • 1990 – Isolation of a human DNA helicase
  • 1992 – Isolation of the first reported mitochondrial DNA helicase
  • 1996 – Report of the discovery of the first purified chloroplast DNA helicase from the pea
  • 2002 – Isolation and characterization of the first biochemically active malarial parasite DNA helicase – Plasmodium cynomolgi.

    Structural features

The common function of helicases accounts for the fact that they display a certain degree of amino acid sequence homology; they all possess sequence motifs located in the interior of their primary structure, involved in ATP binding, ATP hydrolysis and translocation along the nucleic acid substrate. The variable portion of the amino acid sequence is related to the specific features of each helicase.
The presence of these helicase motifs allows putative helicase activity to be attributed to a given protein, but does not necessarily confirm it as an active helicase. Conserved motifs do, however, support an evolutionary homology among enzymes. Based on these helicase motifs, a number of helicase superfamilies have been distinguished.

Superfamilies

Helicases are classified in 6 groups based on their shared sequence motifs. Helicases not forming a ring structure are in superfamilies 1 and 2, and ring-forming helicases form part of superfamilies 3 to 6. Helicases are also classified as α or β depending on if they work with single or double-strand DNA; α helicases work with single-strand DNA and β helicases work with double-strand DNA. They are also classified by translocation polarity. If translocation occurs 3'-5' the helicase is type A; if translocation occurs 5'-3' it is type B.
  • Superfamily 1 : This superfamily can be further subdivided into SF1A and SF1B helicases. In this group helicases can have either 3'-5' or 5'-3' translocation polarity. The most known SF1A helicases are Rep and UvrD in gram-negative bacteria and PcrA helicase from gram-positive bacteria. The most known Helicases in the SF1B group are RecD and Dda helicases. They have a RecA-like-fold core.
  • Superfamily 2 : This is the largest group of helicases that are involved in varied cellular processes. They are characterized by the presence of nine conserved motifs: Q, I, Ia, Ib, and II through VI. This group is mainly composed of DEAD-box RNA helicases. Some other helicases included in SF2 are the RecQ-like family and the Snf2-like enzymes. Most of the SF2 helicases are type A with a few exceptions such as the XPD family. They have a RecA-like-fold core.
  • Superfamily 3 : Superfamily 3 consists of AAA+ helicases encoded mainly by small DNA viruses and some large nucleocytoplasmic DNA viruses. They have a 3'-5' translocation directionality, meaning that they are all type A helicases. The most known SF3 helicase is the papilloma virus E1 helicase.
  • Superfamily 4 : All SF4 family helicases have a type B polarity. They have a RecA fold. The most studied SF4 helicase is gp4 from bacteriophage T7.
  • Superfamily 5 : Rho proteins conform the SF5 group. They have a RecA fold.
  • Superfamily 6 : They contain the core AAA+ that is not included in the SF3 classification. Some proteins in the SF6 group are: mini chromosome maintenance MCM, RuvB, RuvA, and RuvC.
All helicases are members of a P-loop, or Walker motif-containing family.