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Class of enzymes to unpack an organism's genes

Helicases are a class of enzymes thought to be 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 acid phosphodiester backbone, separating two hybridized nucleic acid strands (hence helic + ase), using energy 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 of viral nucleic acids during infection and fulfill an immunological function. A helicase is an enzyme that plays a crucial role in the DNA replication and repair processes. Its primary function is to unwind the double stranded DNA molecule by breaking the hydrogen bonds between the complementary base pairs, allowing the DNA strands to separate. This creates a replication fork, which serves as a template for synthesizing new DNA strands. Helicase is an essential component of cellular mechanisms that ensures accurate DNA replication and maintenance of genetic information. DNA helicase catalyzes regression. RecG and the enzyme PriA work together to rewind duplex DNA, creating a Holliday junction. RecG releases bound proteins and the PriA helicase facilitates DNA reloading to resume DNA replication. RecG replaces the single strand binding protein (SSB), which regulates the helicase fork loading sites during fork regression. The SSB protein interacts with DNA helicases PriA and RecG to recover stalled DNA replication forks. These enzymes must bind to the SSB helicase to be loaded onto stalled forks. Thermal sliding and DNA duplex binding are possibly supported by the wedge domain of RecG's association with the SSB linker. In a regression reaction facilitated by RecG and ATPHollidayjunctions are created for later processing.

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.

Helicases are classified in 6 groups (superfamilies) 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. The most known SF1A helicases are Rep and UvrD in gram negative bacteria and PcrA helicase from gram positive bacteria. They have a 3’ 5’ translocation directionality, meaning that they are all type A helicases. These functions assist in prevention of apoptosis, resulting in cortical size regulation, as well as a contribution to the survival of hippocampal and cortical structures, affecting memory and learning. Mutations have been found throughout the ATRX protein, with over 90% of them being located in the zinc finger and helicase domains. Mutations of ATRX can result in X linked alpha thalassaemia mental retardation (ATR X syndrome). Inherited point mutations in XPD helicase have been shown to be associated with accelerated aging disorders such as Cockayne syndrome (CS) and trichothiodystrophy (TTD). Cockayne syndrome and trichothiodystrophy are both developmental disorders involving sensitivity to UV light and premature aging, and Cockayne syndrome exhibits severe mental retardation from the time of birth. As part of this complex, it facilitates nucleotide excision repair by unwinding DNA. Deficiencies and/or mutations in RecQ family helicases display aberrant genetic recombination and/or DNA replication, which leads to chromosomal instability and an overall decreased ability to proliferate. Bloom syndrome is characterized by a predisposition to cancer with early onset, with a mean age of onset of 24 years. Cells of Bloom syndrome patients show a high frequency of reciprocal exchange between sister chromatids (SCEs) and excessive chromosomal damage. There is evidence to suggest that BLM plays a role in rescuing disrupted DNA replication at replication forks. Cells of Werner syndrome patients exhibit a reduced reproductive lifespan with chromosomal breaks and translocations, as well as large deletions of chromosomal components, causing genomic instability. Chromosomal rearrangements causing genomic instability are found in the cells of Rothmund Thomson syndrome patients. RecQ is a family of DNA helicase enzymes that are found in various organisms including bacteria, archaea, and eukaryotes (like humans). These enzymes play important roles in DNA metabolism during DNA replication, recombination, and repair. There are five known RecQ helicase proteins in humans: RecQ1, BLM, WRN, RecQ4, and RecQ5. Mutations in some of these genes are associated with genetic disorders. For instance, mutations in the BLM gene cause Bloom syndrome, which is characterized by increased cancer risk and other health issues. Mutations in the WRN gene lead to Werner syndrome, a condition characterized by premature aging and an increased risk of age related diseases. RecQ helicases are crucial for maintaining genomic stability and integrity. They help prevent the accumulation of genetic abnormalities that can lead to diseases like cancer. Genome integrity depends on the RecQ DNA helicase family, which includes DNA repair, recombination, replication, and transcription processes. Genome instability and early aging are conditions that arise from mutations in human RecQ helicases. RecQ helicase Sgs1 is missing in yeast cells, making them useful models for comprehending human cell abnormalities and the RecQ helicase function. The RecQ helicase family member, RECQ1, is connected to a small number of uncommon genetic cancer disorders in individuals. It participates in transcription, the cell cycle, and DNA repair. According to recent research, missense mutations in the RECQ1 gene may play a role in the development of familial breast cancer. DNA helicases are frequently attracted to regions of DNA damage and are essential for cellular DNA replication, recombination, repair, and transcription. Chemical manipulation of their molecular processes can change the rate at which cancer cells divide, as well as, the efficiency of transactions and cellular homeostasis. Small molecule induced entrapment of DNA helicases, a type of DNA metabolic protein, may have deleterious consequences on rapidly proliferating cancer cells, which could be effective in cancer treatment. During meiosis DNA double strand breaks and other DNA damages in a chromatid are repaired by homologous recombination using either the sister chromatid or a homologous non sister chromatid as template. This repair can result in a crossover (CO) or, more frequently, a non crossover (NCO) recombinant. In the yeast Schizosaccharomyces pombe the FANCM family DNA helicase FmI1 directs NCO recombination formation during meiosis. The RecQ type helicase Rqh1 also directs NCO meiotic recombination. These helicases, through their ability to unwind D loop intermediates, promote NCO recombination by the process of synthesis dependent strand annealing. In the plant Arabidopsis thaliana, FANCM helicase promotes NCO and antagonizes the formation of CO recombinants. Another helicase, RECQ4A/B, also independently reduces COs. It was suggested that COs are restricted because of the long term costs of CO recombination, that is, the breaking up of favorable genetic combinations of alleles built up by past natural selection. Defective RNA helicases have been linked to cancers, infectious diseases and neuro degenerative disorders. All the eukaryotic RNA helicases that have been identified up to date are non ring forming and are part of SF1 and SF2. On the other hand, ring forming RNA helicases have been found in bacteria and viruses. RNA helicases that do exhibit unwinding activity have been characterized by at least two different mechanisms: canonical duplex unwinding and local strand separation. Canonical duplex unwinding is the stepwise directional separation of a duplex strand, as described above, for DNA unwinding. However, local strand separation occurs by a process wherein the helicase enzyme is loaded at any place along the duplex. This is usually aided by a single strand region of the RNA, and the loading of the enzyme is accompanied with ATP binding. Once the helicase and ATP are bound, local strand separation occurs, which requires binding of ATP but not the actual process of ATP hydrolysis. Presented with fewer base pairs the duplex then dissociates without further assistance from the enzyme. This mode of unwinding is used by the DEAD/DEAH box helicases. An RNA helicase database is currently available online that contains a comprehensive list of RNA helicases with information such as sequence, structure, and biochemical and cellular functions.

Various methods are used to measure helicase activity in vitro. These methods range from assays that are qualitative (assays that usually entail results that do not involve values or measurements) to quantitative (assays with numerical results that can be utilized in statistical and numerical analysis). In 1982–1983, the first direct biochemical assay was developed for measuring helicase activity. This method was called a "strand displacement assay". Strand displacement assay involves the radiolabeling of DNA duplexes. Following helicase treatment, the single strand DNA is visually detected as separate from the double strand DNA by non denaturing PAGE electrophoresis. Following detection of the single strand DNA, the amount of radioactive tag that is on the single strand DNA is quantified to give a numerical value for the amount of double strand DNA unwinding. The strand displacement assay is acceptable for qualitative analysis, its inability to display results for more than a single time point, its time consumption, and its dependence on radioactive compounds for labeling warranted the need for development of diagnostics that can monitor helicase activity in real time. Other methods were later developed that incorporated some, if not all of the following: high throughput mechanics, the use of non radioactive nucleotide labeling, faster reaction time/less time consumption, real time monitoring of helicase activity (using kinetic measurement instead of endpoint/single point analysis). These methodologies include: "a rapid quench flow method, fluorescence based assays, filtration assays, a scintillation proximity assay, a time resolved fluorescence resonance energy transfer assay, an assay based on flashplate technology, homogenous time resolved fluorescence quenching assays, and electrochemiluminescence based helicase assays". With the use of specialized mathematical equations, some of these assays can be utilized to determine how many base paired nucleotides a helicase can break per hydrolysis of 1 ATP molecule. Commercially available diagnostic kits are also available. One such kit is the "Trupoint" diagnostic assay from PerkinElmer, Inc. This assay is a time resolved fluorescence quenching assay that utilizes the PerkinElmer "SignalClimb" technology that is based on two labels that bind in close proximity to one another but on opposite DNA strands. One label is a fluorescent lanthanide chelate, which serves as the label that is monitored through an adequate 96/384 well plate reader. The other label is an organic quencher molecule. The basis of this assay is the "quenching" or repressing of the lanthanide chelate signal by the organic quencher molecule when the two are in close proximity – as they would be when the DNA duplex is in its native state. Upon helicase activity on the duplex, the quencher and lanthanide labels get separated as the DNA is unwound. This loss in proximity negates the quenchers ability to repress the lanthanide signal, causing a detectable increase in fluorescence that is representative of the amount of unwound DNA and can be used as a quantifiable measurement of helicase activity. The execution and use of single molecule fluorescence imaging techniques, focusing on methods that include optical trapping in conjunction with epifluorescent imaging, and also surface immobilization in conjunction with total internal reflection fluorescence visualization. Combined with microchannel flow cells and microfluidic control, allow individual fluorescently labeled protein and DNA molecules to be imaged and tracked, affording measurement of DNA unwinding and translocation at single molecule resolution.

Helicase polarity, which is also deemed "directionality", is defined as the direction (characterized as 5'→3' or 3'→5') of helicase movement on the DNA/RNA single strand along which it is moving. This determination of polarity is vital in f. ex. determining whether the tested helicase attaches to the DNA leading strand, or the DNA lagging strand. To characterize this helicase feature, a partially duplex DNA is used as the substrate that has a central single strand DNA region with different lengths of duplex regions of DNA (one short region that runs 5'→3' and one longer region that runs 3'→5') on both sides of this region. Once the helicase is added to that central single strand region, the polarity is determined by characterization on the newly formed single strand DNA.