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110-450: In E. coli DnaG associates through noncovalent interactions with bacterial replicative helicase DnaB to perform its primase activity, with three DnaG primase proteins associating with each DnaB helicase to form the primosome . Primases tend to initiate synthesis at specific three nucleotide sequences on single-stranded DNA (ssDNA) templates and for E. coli DnaG the sequence is 5'-CTG-3'. DnaG contains three separate protein domains :

220-514: 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

330-520: A "locking" in repair mode. This could cause the helicase to cut DNA segments meant for transcription. Although current evidence points to a defect in the XPD helicase resulting in a loss of flexibility in the protein in cases of Cockayne syndrome, it is still unclear how this protein structure leads to the symptoms described in Cockayne syndrome. In xeroderma pigmentosa, the XPD helicase mutation exists at

440-447: A 3' hydroxyl to form a phosphodiester bond with the next nucleotide. The relatively small number of primase inhibitors likely reflects the inherent difficulty of primase assays rather than a lack of potential binding sites on the enzyme. The short length of products synthesized and the generally slow rate of the enzyme compared to other replication enzymes make developing high-throughput screening (HTS) approaches more difficult. Despite

550-505: A 3-basepair DNA sequence to generate 3-finger, 4-, 5-, or 6-finger arrays that recognize target sites ranging from 9 basepairs to 18 basepairs in length. Another method uses 2-finger modules to generate zinc finger arrays with up to six individual zinc fingers. The Barbas Laboratory of The Scripps Research Institute used phage display to develop and characterize zinc finger domains that recognize most DNA triplet sequences while another group isolated and characterized individual fingers from

660-606: 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

770-515: A bacterial two-hybrid system and has been dubbed "OPEN" by its creators. This system combines pre-selected pools of individual zinc fingers that were each selected to bind a given triplet and then utilizes a second round of selection to obtain 3-finger arrays capable of binding a desired 9-bp sequence. This system was developed by the Zinc Finger Consortium as an alternative to commercial sources of engineered zinc finger arrays. It

880-421: 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. Zinc finger A zinc finger is a small protein structural motif that

990-499: A contribution to the survival of hippocampal and cortical structures, affecting memory and learning. This helicase is located on the X chromosome (Xq13.1-q21.1), in the pericentromeric heterochromatin and binds to heterochromatin protein 1 . Studies have shown that ATRX plays a role in rDNA methylation and is essential for embryonic development. Mutations have been found throughout the ATRX protein, with over 90% of them being located in

1100-435: A double-strand break to a desired genomic locus can be used to introduce frame-shift mutations into the coding sequence of a gene due to the error-prone nature of the non-homologous DNA repair pathway. If a homologous DNA "donor sequence" is also used then the genomic locus can be converted to a defined sequence via the homology directed repair pathway. An ongoing clinical trial is evaluating Zinc finger nucleases that disrupt

1210-457: 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

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1320-510: A few. Zinc-binding motifs are stable structures, and they rarely undergo conformational changes upon binding their target. Initially, the term zinc finger was used solely to describe DNA-binding motif found in Xenopus laevis ; however, it is now used to refer to any number of structures related by their coordination of a zinc ion. In general, zinc fingers coordinate zinc ions with a combination of cysteine and histidine residues. Originally,

1430-513: A given DNA target from a large pool of partially randomized zinc finger arrays. This technique is difficult to use on more than a single zinc finger at a time, so a multi-step process that generated a completely optimized 3-finger array by adding and optimizing a single zinc finger at a time was developed. More recent efforts have utilized yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells. A promising new method to select novel 3-finger zinc finger arrays utilizes

1540-667: A given gene can be used to alter the transcription of that gene. Fusions between engineered zinc finger arrays and protein domains that cleave or otherwise modify DNA can also be used to target those activities to desired genomic loci. The most common applications for engineered zinc finger arrays include zinc finger transcription factors and zinc finger nucleases , but other applications have also been described. Typical engineered zinc finger arrays have between 3 and 6 individual zinc finger motifs and bind target sites ranging from 9 basepairs to 18 basepairs in length. Arrays with 6 zinc finger motifs are particularly attractive because they bind

1650-525: A loop and a second β-hairpin of varying length and conformation can be present between the N-terminal β-hairpin and the C-terminal α-helix. These fingers are present in a diverse group of proteins that frequently do not share sequence or functional similarity with each other. The best-characterized proteins containing treble-clef zinc fingers are the nuclear hormone receptors . The zinc ribbon fold

1760-551: A number of types of zinc fingers, each with a unique three-dimensional architecture. A particular zinc finger protein's class is determined by its three-dimensional structure, but it can also be recognized based on the primary structure of the protein or the identity of the ligands coordinating the zinc ion. In spite of the large variety of these proteins, however, the vast majority typically function as interaction modules that bind DNA , RNA , proteins, or other small, useful molecules, and variations in structure serve primarily to alter

1870-426: A phosphodiester bond via dehydration synthesis between the 3' hydroxyl of the nucleotide in the initiation site and the α-phosphate of the nucleotide in the elongation site. This reaction results in a dinucleotide and breaking of the bond between the α and β phosphorus, releasing pyrophosphate. This reaction is irreversible because the pyrophosphate that is formed is hydrolyzed into two inorganic phosphate molecules by

1980-467: A reduced reproductive lifespan with chromosomal breaks and translocations, as well as large deletions of chromosomal components, causing genomic instability. Rothmund-Thomson syndrome, also known as poikiloderma congenitale , is characterized by premature aging, skin and skeletal abnormalities, rash, poikiloderma , juvenile cataracts, and a predisposition to cancers such as osteosarcomas. Chromosomal rearrangements causing genomic instability are found in

2090-439: 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

2200-450: A role in recognizing sequence specific DNA binding sites. The central domain (residues 111–433) displays RNA polymerase activities, and is the site of RNA primer synthesis. The C-terminal domain (residues 434–581) is responsible for the noncovalent binding of DnaG to the DnaB helicase protein. The zinc-binding domain, the domain responsible for recognizing sequence specific DNA binding sites,

2310-399: A role in sequence recognition. In addition, the hydrophobic surface of the β sheet, as well as the basic residues which are clustered primarily on one edge of the sheet, serve to attract single stranded DNA, further facilitating DNA binding. Based on previous studies of DNA binding by DNA Primases, it is thought that DNA binds to the zinc-binding domain across the surface of the β sheet, with

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2420-485: A similar manner, as the residues important for binding DNA in RPA occur in structurally equivalent positions in B. stearothermophilus . As its name suggests, the RNA polymerase domain (RNAP) of DnaG is responsible for synthesizing the RNA primers on the single stranded DNA. In-vivo, DnaG is able to synthesis primer fragments of up to 60 nucleotides, but in-vivo primer fragments are limited to approximately 11 nucleotides. During

2530-466: A specific sequence is an area of active research, and zinc finger nucleases and zinc finger transcription factors are two of the most important applications of this to be realized to date. Zinc fingers were first identified in a study of transcription in the African clawed frog , Xenopus laevis in the laboratory of Aaron Klug . A study of the transcription of a particular RNA sequence revealed that

2640-513: A target site that is long enough to have a good chance of being unique in a mammalian genome. Engineered zinc finger arrays are often fused to a DNA cleavage domain (usually the cleavage domain of FokI ) to generate zinc finger nucleases . Such zinc finger-FokI fusions have become useful reagents for manipulating genomes of many higher organisms including Drosophila melanogaster , Caenorhabditis elegans , tobacco , corn , zebrafish , various types of mammalian cells, and rats . Targeting

2750-554: A variety of functions such as binding RNA and mediating protein-protein interactions, but is best known for its role in sequence-specific DNA-binding proteins such as Zif268 (Egr1). In such proteins, individual zinc finger domains typically occur as tandem repeats with two, three, or more fingers comprising the DNA-binding domain of the protein. These tandem arrays can bind in the major groove of DNA and are typically spaced at 3-bp intervals. The α-helix of each domain (often called

2860-466: A zinc binding domain, an RNA polymerase domain, and a DnaB helicase binding domain. There are several bacteria that use the DNA primase DnaG. A few organisms that have DnaG as their DNA primase are Escherichia coli ( E. coli ), Bacillus stearothermophilus , and Mycobacterium tuberculosis (MTB). E. coli DnaG has a molecular weight of 60 kilodaltons (kDa) and contains 581 amino acids . DnaG catalyzes

2970-451: Is a history of helicase discovery: 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

3080-406: Is achieved through the lowering of the activation barrier ( B {\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

3190-473: 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

3300-428: 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

3410-682: Is best suited for most applications. The most straightforward method to generate new zinc finger arrays is to combine smaller zinc finger "modules" of known specificity. The structure of the zinc finger protein Zif268 bound to DNA described by Pavletich and Pabo in their 1991 publication has been key to much of this work and describes the concept of obtaining fingers for each of the 64 possible base pair triplets and then mixing and matching these fingers to design proteins with any desired sequence specificity. The most common modular assembly process involves combining separate zinc fingers that can each recognize

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3520-450: Is characterised by two beta-hairpins forming two structurally similar zinc-binding sub-sites. The canonical members of this class contain a binuclear zinc cluster in which two zinc ions are bound by six cysteine residues. These zinc fingers can be found in several transcription factors including the yeast Gal4 protein. The zinc finger antiviral protein ( ZAP ) binds to the CpG site. It

3630-408: Is characterized by the coordination of one or more zinc ions (Zn ) which stabilizes the fold. It was originally coined to describe the finger-like appearance of a hypothesized structure from the African clawed frog ( Xenopus laevis ) transcription factor IIIA . However, it has been found to encompass a wide variety of differing protein structures in eukaryotic cells. Xenopus laevis TFIIIA

3740-460: Is close in proximity to, but does not make contact with, the HBD of DnaG. Mutation of Tyr88 inhibits the formation of the N-terminal domain helical bundle of DnaB, interrupting the contacts with the HBD of DnaG. The hexameric structure of DnaB is really a trimer of dimers. Both Ile119 and Ile125 are buried in the N-terminal domain dimer interface of DnaB and mutation of these residues inhibits formation of

3850-546: 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

3960-457: Is conserved across all viral, bacteriophage, prokaryotic and eukaryotic DNA primases. The primase zinc-binding domain is part of the subfamily of zinc-binding domains known as the zinc ribbon . Zinc ribbon domains are characterized by two β-hairpin loops which form the zinc-binding domain. Typically, zinc ribbon domains are thought to lack α-helices , distinguishing them from other zinc-binding domains. However, in 2000 DnaG's zinc-binding domain

4070-432: Is evidence to suggest that BLM plays a role in rescuing disrupted DNA replication at replication forks. Werner syndrome is a disorder of premature aging, with symptoms including early onset of atherosclerosis and osteoporosis and other age related diseases, a high occurrence of sarcoma, and death often occurring from myocardial infarction or cancer in the 4th to 6th decade of life. Cells of Werner syndrome patients exhibit

4180-477: Is made up in part of the toprim fold , a fold that has been observed in many metal-binding phosphotransfer proteins. The central domain and the N-terminal domain form a shallow cleft, which makes up the active site of the RNA chain elongation in DnaG. The opening of the cleft is lined by several highly conserved basic residues: Arg146, Arg221, and Lys229. These residues are part of the electrostatically positive ridge of

4290-513: 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

4400-542: Is the Boltzmann constant and T {\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

4510-487: 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

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4620-690: 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

4730-542: Is type B. All helicases are members of a P-loop, or Walker motif -containing family. The ATRX gene encodes the ATP-dependent helicase, ATRX (also known as XH2 and XNP) of the SNF2 subgroup family, that is thought to be responsible for functions such as chromatin remodeling, gene regulation, and DNA methylation. These functions assist in prevention of apoptosis, resulting in cortical size regulation, as well as

4840-408: Is used in mammals for antiviral defense. Various protein engineering techniques can be used to alter the DNA-binding specificity of zinc fingers and tandem repeats of such engineered zinc fingers can be used to target desired genomic DNA sequences. Fusing a second protein domain such as a transcriptional activator or repressor to an array of engineered zinc fingers that bind near the promoter of

4950-534: 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

5060-414: The "recognition helix") can make sequence-specific contacts to DNA bases; residues from a single recognition helix can contact four or more bases to yield an overlapping pattern of contacts with adjacent zinc fingers. This fold group is defined by two short β-strands connected by a turn (zinc knuckle) followed by a short helix or loop and resembles the classical Cys 2 His 2 motif with a large portion of

5170-552: 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

5280-552: The CCR5 gene in CD4 human T-cells as a potential treatment for HIV/AIDS . The majority of engineered zinc finger arrays are based on the zinc finger domain of the murine transcription factor Zif268, although some groups have used zinc finger arrays based on the human transcription factor SP1. Zif268 has three individual zinc finger motifs that collectively bind a 9 bp sequence with high affinity. The structure of this protein bound to DNA

5390-482: The Cys 2 His 2 -like (the "classic zinc finger"), treble clef, and zinc ribbon. The following table shows the different structures and their key features: The Cys 2 His 2 -like fold group (C2H2) is by far the best-characterized class of zinc fingers, and is common in mammalian transcription factors. Such domains adopt a simple ββα fold and have the amino acid sequence motif : This class of zinc fingers can have

5500-481: The DNA leading strand or Okazaki fragments for the lagging strand. The rate limiting step of the primer synthesis occurs after NTP binding but before or during dinucleotide synthesis. The E. Coli DnaG primase is a 581 residue monomeric protein with three functional domains, according to proteolysis studies. There is an N-terminal Zinc-binding domain (residues 1–110) where a zinc ion is tetrahedrally coordinated between one histidine and three cysteine residues, which plays

5610-413: The N-terminal subdomain. It is this ridge that interacts with the ssDNA and helps guide it into the cleft, which consists of the metal binding center of the toprim motif on the central subdomain, and the conserved primase motifs of the N-terminal domain. The metal binding site of the toprim domain is where the primer is synthesized. The RNA:DNA duplex then exits through another basic depression. Unlike both

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5720-432: The RNA primers is not random, suggesting that they are placed on specific DNA sequences. Indeed, other DNA primases have been shown to recognize triplet sequences; the specific sequence recognized by B. stearothermophilus has not yet been identified. It has been shown that if the cystine residues that coordinate the zinc ion are mutated, the DNA primase stops functioning. This indicates that the zinc-binding domain does play

5830-510: 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,

5940-586: The XPD helicase that helps form this complex and contributes to its function causes the sensitivity to sunlight seen in all three diseases, as well as the increased risk of cancer seen in XP and premature aging seen in trichothiodystrophy and Cockayne syndrome. XPD helicase mutations leading to trichothiodystrophy are found throughout the protein in various locations involved in protein-protein interactions. This mutation results in an unstable protein due to its inability to form stabilizing interactions with other proteins at

6050-784: 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,

6160-408: The autosomal recessive diseases Bloom syndrome (BS), Rothmund–Thomson syndrome (RTS), and Werner syndrome (WS), respectively. 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

6270-405: The binding specificity of a particular protein. Since their original discovery and the elucidation of their structure, these interaction modules have proven ubiquitous in the biological world and may be found in 3% of the genes of the human genome. In addition, zinc fingers have become extremely useful in various therapeutic and research capacities. Engineering zinc fingers to have an affinity for

6380-461: The binding strength of a small transcription factor (transcription factor IIIA; TFIIIA) was due to the presence of zinc-coordinating finger-like structures. Amino acid sequencing of TFIIIA revealed nine tandem sequences of 30 amino acids, including two invariant pairs of cysteine and histidine residues. Extended x-ray absorption fine structure confirmed the identity of the zinc ligands: two cysteines and two histidines. The DNA-binding loop formed by

6490-458: The canonical pattern of interactions of zinc fingers with DNA. The binding of zinc finger is found to be distinct from many other DNA-binding proteins that bind DNA through the 2-fold symmetry of the double helix, instead zinc fingers are linked linearly in tandem to bind nucleic acid sequences of varying lengths. Zinc fingers often bind to a sequence of DNA known as the GC box . The modular nature of

6600-477: 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

6710-402: 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

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6820-407: The compounds known to inhibit primases are nucleotide analogs such as AraATP (see Vidarabine ) and 2-fluoro-AraATP. These compounds will often be used as substrates by the primase, but once incorporated synthesis or elongation can no longer occur. For example, E. coli DnaG will use 2',3'-dideoxynucleoside 5'-triphosphates (ddNTPs) as substrates, which act as chain terminators due to the lack of

6930-610: The coordination of these ligands by zinc were thought to resemble fingers, hence the name. This was followed soon thereafter by the discovery of the Krüppel factor in Drosophila by the Schuh team in 1986. More recent work in the characterization of proteins in various organisms has revealed the importance of zinc ions in polypeptide stabilization. The crystal structures of zinc finger-DNA complexes solved in 1991 and 1993 revealed

7040-462: The difficulties, there are several known inhibitors of DnaG that are not NTP analogues. Doxorubicin and suramin are both DNA and NTP competitive inhibitors of Mycobacterium Tuberculosis DnaG. Suramin is also known to inhibit eukaryotic DNA primase by competing with GTP, so suramin is likely to inhibit DnaG via a similar mechanism. Helicase Helicases are a class of enzymes thought to be vital to all organisms . Their main function

7150-449: The dinucleotide to occur, oligonucleotide must be moved so that the 3' NTP is transferred from the elongation site to the initiation site, allowing for another NTP to bind to the elongation site and attach to the 3' hydroxyl of the oligonucleotide. Once an oligonucleotide of appropriate length has been synthesized from the elongation step of primer synthesis, DnaG transfers the newly synthesized primer to DNA polymerase III for it to synthesize

7260-515: 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

7370-472: 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

7480-419: The enzyme inorganic pyrophosphatase . This dinucleotide synthesis reaction is the same reaction as any other enzyme that catalyzes the formation of DNA or RNA ( DNA Polymerase , RNA Polymerase ), therefore DnaG must always synthesize oligonucleotides in the 5' to 3' direction. In E. coli , primers begin with a triphosphate adenine-guanine (pppAG) dinucleotide at the 5' end. In order for further elongation of

7590-413: The finger-like folds . They were first identified as a DNA-binding motif in transcription factor TFIIIA from Xenopus laevis (African clawed frog), however they are now recognised to bind DNA, RNA, protein, and/or lipid substrates . Their binding properties depend on the amino acid sequence of the finger domains and on the linker between fingers, as well as on the higher-order structures and

7700-563: The first direct biochemical assay was developed for measuring helicase activity. This method was called a "strand displacement assay". 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,

7810-407: The genome and suppress inappropriate recombination. 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. Mutations in RecQ family helicases BLM, RECQL4 , and WRN, which play a role in regulating homologous recombination, have been shown to result in

7920-466: The helicase superfamilies except for SF6. 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. However, not all RNA helicases exhibit helicase activity as defined by enzymatic function, i.e., proteins of the Swi/Snf family. Although these proteins carry

8030-616: The helix and β-hairpin truncated. The retroviral nucleocapsid (NC) protein from HIV and other related retroviruses are examples of proteins possessing these motifs. The gag-knuckle zinc finger in the HIV NC protein is the target of a class of drugs known as zinc finger inhibitors . The treble-clef motif consists of a β-hairpin at the N-terminus and an α-helix at the C-terminus that each contribute two ligands for zinc binding, although

8140-605: The hexameric structure and thus the interaction with DnaG. One other residue that has been identified as playing a crucial role in the interaction of DnaB and DnaG is Glu15. Mutation of Glu15 does not disrupt the formation of the DnaB, DnaG complex, but instead plays a role in modulating the length of primers synthesized by DnaG. Inhibitors of DNA primases are valuable compounds for the elucidation of biochemical pathways and key interactions, but they are also of interest as lead compounds to design drugs against bacterial diseases. Most of

8250-460: The human genome. A potential drawback with modular assembly in general is that specificities of individual zinc finger can overlap and can depend on the context of the surrounding zinc fingers and DNA. A recent study demonstrated that a high proportion of 3-finger zinc finger arrays generated by modular assembly fail to bind their intended target with sufficient affinity in a bacterial two-hybrid assay and fail to function as zinc finger nucleases , but

8360-456: The hydrophobic inner surface of the β sheet which is packed against the second and third α helices. The outer surface of the β sheet also has many conserved hydrophobic and basic residues. These residues are Lys30, Arg34, Lys46, Pro48, Lys56, Ile58, His60 and Phe62. It is thought that the function of the zinc binding domain is for sequence specific DNA recognition. DNA primases make RNA primers which are then used for DNA synthesis. The placement of

8470-733: 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

8580-586: The mediation of antiviral immune response because they can identify foreign RNAs in vertebrates. About 80% of all viruses are RNA viruses and they contain their own RNA helicases. Defective RNA helicases have been linked to cancers, infectious diseases and neuro-degenerative disorders. Some neurological disorders associated with defective RNA helicases are: amyotrophic lateral sclerosis , spinal muscular atrophy , spinocerebellar ataxia type-2 , Alzheimer disease , and lethal congenital contracture syndrome . RNA helicases and DNA helicases can be found together in all

8690-798: The mutation of ATRX gene causes the downregulation of gene expression, such as the alpha-globin genes. It is still unknown what causes the expression of the various characteristics of ATR-X in different patients. XPD (Xeroderma pigmentosum factor D, also known as protein ERCC2) is a 5'-3', Superfamily II, ATP-dependent helicase containing iron-sulphur cluster domains. 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

8800-425: The number and order of these residues was used to classify different types of zinc fingers ( e.g., Cys 2 His 2 , Cys 4 , and Cys 6 ). More recently, a more systematic method has been used to classify zinc finger proteins instead. This method classifies zinc finger proteins into "fold groups" based on the overall shape of the protein backbone in the folded domain. The most common "fold groups" of zinc fingers are

8910-694: The number of fingers. Znf domains are often found in clusters, where fingers can have different binding specificities. Znf motifs occur in several unrelated protein superfamilies , varying in both sequence and structure. They display considerable versatility in binding modes, even between members of the same class (e.g., some bind DNA, others protein), suggesting that Znf motifs are stable scaffolds that have evolved specialised functions. For example, Znf-containing proteins function in gene transcription, translation, mRNA trafficking, cytoskeleton organization, epithelial development, cell adhesion , protein folding, chromatin remodeling, and zinc sensing, to name but

9020-458: 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

9130-406: The points of mutations. This, in turn, destabilizes the entire TFIIH complex, which leads to defects with transcription and repair mechanisms of the cell. It has been suggested that XPD helicase mutations leading to Cockayne syndrome could be the result of mutations within XPD, causing rigidity of the protein and subsequent inability to switch from repair functions to transcription functions due to

9240-458: The primase and templates. The two NTP binding sites on DnaG are referred to as the initiation site and elongation site. The initiation site is the site at which the NTP to be incorporated at the 5' end of the primer binds. The elongation site binds the NTP that is added to the 3' end of the primer. Once two nucleotides are bound to the primase, DnaG catalyzes the formation of a dinucleotide by forming

9350-463: The primase protein (DnaG). Prior to the binding of any NTPs to form the RNA primer, the ssDNA template sequence binds to DnaG. The ssDNA contains a three nucleotide recognition sequence that recruits NTPs based on Watson-Crick base pairing . After binding DNA, DnaG must bind two NTPs in order to generate an enzyme-DNA-NTP-NTP quaternary complex. The Michaelis constant's (km) for the NTPs vary depending on

9460-673: 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 favourable genetic combinations of alleles built up by past natural selection . RNA helicases are essential for most processes of RNA metabolism such as ribosome biogenesis, pre-mRNA splicing, and translation initiation. They also play an important role in sensing viral RNAs. RNA helicases are involved in

9570-697: The range of 5,000 to 10,000 R.P.M. 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

9680-452: 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

9790-505: 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 V un {\displaystyle V_{\text{un}}} and V trans {\displaystyle V_{\text{trans}}} are approximately equal. These two categories of helicases may also be modeled as mechanisms. In such models,

9900-423: 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 B > k B T {\displaystyle B>k_{\text{B}}T} , where k B {\displaystyle k_{\text{B}}}

10010-401: The second and third β sheet. Cys61 is located on the fifth β sheet, and Cys64 is on the β-hairpin between the fourth and fifth β sheet. These four residues coordinate the zinc ion tetrahedrally. The zinc ion is thought to stabilize the loops between the second and third β sheet as well as the fourth and fifth β sheet. The domain is further stabilized by a number of hydrophobic interactions between

10120-414: 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

10230-517: The site of ATP or DNA binding. This results in a structurally functional helicase able to facilitate transcription, however it inhibits its function in unwinding DNA and DNA repair. The lack of a cell's ability to repair mutations, such as those caused by sun damage, is the cause of the high cancer rate in xeroderma pigmentosa patients. RecQ helicases (3'-5') belong to the Superfamily II group of helicases, which help to maintain stability of

10340-689: The success rate was somewhat higher when sites of the form GNNGNNGNN were targeted. A subsequent study used modular assembly to generate zinc finger nucleases with both 3-finger arrays and 4-finger arrays and observed a much higher success rate with 4-finger arrays. A variant of modular assembly that takes the context of neighboring fingers into account has also been reported and this method tends to yield proteins with improved performance relative to standard modular assembly. Numerous selection methods have been used to generate zinc finger arrays capable of targeting desired sequences. Initial selection efforts utilized phage display to select proteins that bound

10450-503: The synthesis of oligonucleotides in five discrete steps: template binding, nucleoside triphosphate (NTP) binding, initiation, extension to form a primer, and primer transfer to DNA polymerase III. DnaG performs this catalysis near the replication fork that is formed by DnaB helicase during DNA replication. DnaG must be complexed with DnaB in order for it to catalyze the formation of the oligonucleotide primers. The mechanism for primer synthesis by primases involves two NTP binding sites on

10560-422: The synthesis of the lagging strand DnaG synthesizes between 2000 and 3000 primers at a rate of one primer per-second. RNAP domain of DnaG has three subdomains, the N-terminal domain, which has a mixed α and β fold, the central domain consisting of a 5 stranded β sheet and 6 α helices, and finally the C-terminal domain which is made up of a helical bundle consisting of 3 antiparallel α helices. The central domain

10670-422: 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, V un {\displaystyle V_{\text{un}}} is closer to V trans {\displaystyle V_{\text{trans}}} , due to the active helicase ability to directly destabilize

10780-425: The three nucleotides binding across three strands of the β sheet. The positively charged residues in the sheet would be able to form contacts with the phosphates and the aromatic residues would form stacking interactions with the bases. This is the model of DNA binding by the ssDNA-binding domain of replication protein A (RPA) . It is logical to assume that B. stearothermophilus’ zinc-binding domain binds DNA in

10890-562: The time of birth. The XPD helicase mutation has also been implicated in xeroderma pigmentosum (XP), a disorder characterized by sensitivity to UV light and resulting in a several 1000-fold increase in the development of skin cancer. XPD is an essential component of the TFIIH complex, a transcription and repair factor in the cell. As part of this complex, it facilitates nucleotide excision repair by unwinding DNA. TFIIH assists in repairing damaged DNA such as sun damage. A mutation in

11000-605: The two to three DnaG molecules that bind the DnaB hexamer, the C1 subdomains of the HBDs interact with DnaB at its N-terminal domains on the inner surface of the hexamer ring, while the C2 subdomains interact with the N-terminal domains on the outer surface of the hexamer. Three residues in B. stearothermophilus DnaB have been identified as important for formation of the DnaB, DnaG interface. Those residues include Tyr88, Ile119, and Ile125. Tyr88

11110-402: The typical helicase motifs, hydrolize ATP in a nucleic acid-dependent manner, and are built around a helicase core, in general, no unwinding activity is observed. 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

11220-577: The unwinding rate. In passive systems, the rate of unwinding ( V u n {\displaystyle V_{un}} ) is less than the rate of translocation ( V t r a n s {\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. In active helicases, B < k B T {\displaystyle B<k_{\text{B}}T} , where

11330-815: 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. 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

11440-772: The zinc finger and helicase domains. Mutations of ATRX can result in X-linked-alpha-thalassaemia-mental retardation ( ATR-X syndrome ). Various types of mutations found in ATRX have been found to be associated with ATR-X, including most commonly single-base missense mutations, as well as nonsense, frameshift, and deletion mutations. Characteristics of ATR-X include: microcephaly, skeletal and facial abnormalities, mental retardation, genital abnormalities, seizures, limited language use and ability, and alpha-thalassemia. The phenotype seen in ATR-X suggests that

11550-524: The zinc finger motif allows for a large number of combinations of DNA and RNA sequences to be bound with high degree of affinity and specificity, and is therefore ideally suited for engineering protein that can be targeted to and bind specific DNA sequences. In 1994, it was shown that an artificially-constructed three-finger protein can block the expression of an oncogene in a mouse cell line. Zinc fingers fused to various other effector domains, some with therapeutic significance, have since been constructed. Such

11660-419: The zinc-binding domains, and the RNA polymerase domains, the C-terminal domains of DNA primases are not conserved. In prokaryotic primases, the only known function of this domain is to interact with the helicase, DnaB. Thus, this domain is called the helicase binding domain (HBD). The HBD of DnaG consists of two subdomains: a helical bundle , the C1 subdomain, and a helical hairpin, the C2 subdomain. For each of

11770-417: Was crystallized from Bacillus stearothermophilus revealing that the domain consisted of a five stranded antiparallel β sheet adjacent to four α helices and a 3 10 helix on the c-terminal end of the domain. The zinc-binding site of B. stearothermophilus consists of three cysteine residues, Cys40, Cys61, and Cys64, and one histidine residue, His43. Cys40 and His43 are located on the β-hairpin between

11880-740: Was its importance that "the zinc-finger motif" was cited in the Scientific Background to the 2024 Nobel Prize in Chemistry (awarded to David Baker , Demis Hassabis , and John M. Jumper for computational protein design and protein structure prediction). Zinc finger (Znf) domains are relatively small protein motifs that contain multiple finger-like protrusions that make tandem contacts with their target molecule. Some of these domains bind zinc, but many do not, instead binding other metals such as iron, or no metal at all. For example, some family members form salt bridges to stabilise

11990-532: Was originally demonstrated to contain zinc and require the metal for function in 1983, the first such reported zinc requirement for a gene regulatory protein followed soon thereafter by the Krüppel factor in Drosophila . It often appears as a metal-binding domain in multi-domain proteins. Proteins that contain zinc fingers ( zinc finger proteins ) are classified into several different structural families. Unlike many other clearly defined supersecondary structures such as Greek keys or β hairpins , there are

12100-399: Was solved in 1991 and stimulated a great deal of research into engineered zinc finger arrays. In 1994 and 1995, a number of groups used phage display to alter the specificity of a single zinc finger of Zif268. There are two main methods currently used to generate engineered zinc finger arrays, modular assembly, and a bacterial selection system, and there is some debate about which method

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