The Evolution of COVID-19 Diagnostics
Debmalya Barh, Kenneth Lundstrom in COVID-19, 2022
The helicase dependent isothermal DNA amplification technique uses DNA helicase to separate double-stranded DNA and to generate a single-stranded template for hybridization of a primer with subsequent primer extension by a DNA polymerase, resulting in an exponential amplification of a selected DNA target [31]. This method helps to maintain a constant temperature from the beginning to the end of the reaction by eliminating the initial PCR denaturation step [32]. Quidel has developed an isothermal helicase dependent amplification with fluorescence detection of SARS-CoV-2 (https://www.quidel.com/molecular-diagnostics/helicase-dependent-amplification-tests). The technique operates in individual tubes at one temperature reducing the time required to 25 minutes. It also avoids the need for specialized training in molecular techniques.
Transport of mRNA into the Cytoplasm
Alvaro Macieira-Coelho in Molecular Basis of Aging, 2017
Besides “poly(A)”, RNA stem-loop structures, which are present at the 5′- and 3′-terminal untranslated regions of a number of mRNAs, can represent further recognition signals for the mRNA translocation system. These double-stranded RNA regions can serve as binding sites for regulatory proteins (e.g., in the case of ferritin-, poliovirus-, and HIV-1 mRNA) or they can increase the stability of the mRNAs (e.g., in the case of histone mRNAs). The double-stranded parts within the RNA stem loops are unwound by a NE-associated RNA helicase.26 The helicase activity can be determined by measuring the unwinding of in vitro synthesized, partially double-stranded RNA substrates.26 We showed that the enzyme in the NE really represents a helicase and not a RNA-modifying/unwinding activity; the unwound single-stranded RNAs can be rehybridized to form the double-stranded RNA substrate. Therefore the helicase is functionally distinct from RNA-modifying enzymes, which cause unwinding of double-stranded RNAs by transition of adenosine to inosine residues.27
The Premature Aging Characteristics of RecQ Helicases
Shamim I. Ahmad in Aging: Exploring a Complex Phenomenon, 2017
The WRN gene was identified in 1996 through positional cloning and either homozygous or compound heterozygous loss of function mutations are the cause of WS, numbering greater than 80 distinct mutations in the WRN gene [37]. WRN encodes a 160-kDa protein, WRN, of 1432 amino acids that contains a central ATP-dependent 3′–5′ RecQ DNA helicase domain that includes the RQC region, an N-terminal 3′–5′ exonuclease domain, as well as a C-terminal HRDC domain and a nuclear localization signal (NLS). Most of the disease-causing mutations are either stop codons, splice variants, or small indels that promote nonsense-mediated mutant mRNA decay [38] and/or result in a truncated WRN. The truncated forms of WRN protein have lost the C-terminal NLS and therefore cannot reach the nucleus and are subsequently degraded in the cytoplasm [39–41]. There are a few mutations that instead cause amino acid substitutions. Lys125Asn and Lys135Glu both occur in the exonuclease domain, and both of these result in an unstable form of WRN protein that functionally acts as a null mutation. Further, two amino acid substitution mutations, Gly574Arg and Arg637Trp, have been characterized in the central WRN helicase domain and are observed as compound heterozygotes associated with null mutations in WS individuals [42,43]. The Gly574Arg mutation ablates the WRN helicase activity, while Arg637Trp is predicted to inactivate helicase function [44], and the clinical phenotypes of individuals with either of these mutations appear identical to those carrying null mutations [43,44].
Emergence of varicella-zoster virus resistance to acyclovir: epidemiology, prevention, and treatment
Published in Expert Review of Anti-infective Therapy, 2021
Kimiyasu Shiraki, Masaya Takemoto, Tohru Daikoku
Double-stranded DNA needs to be separated into two single strands (replication fork) before DNA synthesis, and complementary strands are synthesized from each DNA strand to produce two new double-stranded DNA molecules during DNA replication (Figure 3). The HP complex is responsible for unwinding viral DNA at the replication fork, separating double-stranded DNA into two single strands, and synthesizing RNA primers (Okazaki fragments) in the lagging strand for DNA synthesis. DNApol initiates complementary DNA synthesis in the two separated DNA strands. The HP complex consists of three proteins: VZVORF55 (helicase), VZVORF6 (primase), and VZVORF52 (cofactor). The helicase unwinds the duplex DNA ahead of the fork and separates the double strand into two single strands. The primase lays down RNA primers that extend the two-subunit DNApol. The HP complex possesses multienzymatic activities, including DNA-dependent ATPase, helicase, and primase activities, all of which are required for the HP complex to function in viral DNA replication.
Coronaviruses
Published in Expert Opinion on Therapeutic Patents, 2021
Claudiu T. Supuran
Spratt et al. [27] discuss in detail the helicase of SARS-CoV-2 (and the closely related viruses), its mechanism of action and inhibition, also presenting the available inhibitors and drug design studies in the scientific and patent literature. Helicases are nucleic acid unwinding enzymes present in many (but not all) viruses, and ultimately, they started to be considered as drug targets of interest. Indeed, pritelivir, a helicase-primase primary sulfonamide inhibitor [30] (Figure 2) was clinically approved in 2020 for the treatment of herpes simplex virus infections in immunocompromised patients with drug resistance to other antivirals [31]. It is interesting to note that pritelivir is also a highly effective inhibitor of the zinc enzyme carbonic anhydrase [30,32], as most primary sulfonamides [33].
Coronavirus helicases: attractive and unique targets of antiviral drug-development and therapeutic patents
Published in Expert Opinion on Therapeutic Patents, 2021
Austin N. Spratt, Fabio Gallazzi, Thomas P. Quinn, Christian L. Lorson, Anders Sönnerborg, Kamal Singh
Helicases are ubiquitous nucleic acid unwinding enzymes. These biological motors couple the chemical energy of nucleotide triphosphate hydrolysis (NTPase) to mechanical energy that translocates through nucleic acids, unwinding the helical structure as it progresses, thus the term ‘helicase.’ Efficient genome replication, recombination and repair require single stranded DNA (ssDNA) or single stranded RNA (ssRNA) as a template that is largely devoid of secondary structures [1]. Helicases in situ generate ssDNA or ssRNA, and due to this crucial role during genome replication, repair and recombination, defects in helicase function can lead to many genetic disorders. Notable examples of helicase-associated disorders include Bloom’s syndrome, Werner’s syndrome, and X-chromosome-linked α-thalassemia [2–9].
Related Knowledge Centers
- Directionality
- DNA
- Enzyme
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- Hydrolysis
- Motor Protein
- Nucleic Acid
- Phosphodiester Bond
- DNA
- Nucleic Acid Double Helix
- Adenosine Triphosphate
- Genome