Instability of Human Mitochondrial DNA, Nuclear Genes and Diseases
Shamim I. Ahmad in Handbook of Mitochondrial Dysfunction, 2019
In 2013 a new enzyme PRIMPOL was described and precharacterized as a protein exhibiting two activities – primase and polymerase with unusual properties: it can by itself start DNA synthesis and is extremely resistant to DNA damage18. PRIMPOL acts both in the nucleus and mitochondrion and seems to play similar role in both compartments – it rescues stalled replication forks by adding dNTP at the site of lesion19. It does not play a role in the initiation of replication. Only one variant in PRIMPOL (4q35.1) (p.Tyr89Asp) was suggested to play role in autosomal dominant high myopia20. Although it was proven that this variant negatively influences processivity of the enzyme21 it is difficult to say whether the proposed phenotype results from alteration of nuclear, mitochondrial or both functions. Moreover, the involvement of PRIMPOL mutation in high myopia was later questioned22. The RNA polymerase involved in transcription, playing the role of primase in mtDNA replication will be presented later.
Introduction to Molecular Biology
Martin G. Pomper, Juri G. Gelovani, Benjamin Tsui, Kathleen Gabrielson, Richard Wahl, S. Sam Gambhir, Jeff Bulte, Raymond Gibson, William C. Eckelman in Molecular Imaging in Oncology, 2008
After the unwinding of the double helix, a new DNA strand is synthesized in the 5′ to 3′ direction for each parental strand by an enzyme called DNA polymerase. The synthesis of the new strand at the replication fork is bidirectional (Fig. 9), with two different DNA polymerases in function. One is for the leading strand and the other is for the lagging strand. In the leading strand, the DNA polymerase catalyzes the formation of a phosphodiester bond between the 5′-phosphate on an incoming nucleotide and the free 3′-hydroxyl group on the growing polynucleotide. Thus, the growing DNA fragment is synthesized by complementary base pairing with the parental DNA template. By this mechanism, the DNA polymerase moves along the template and adds nucleotides in the 5′ → 3′ direction. This is true for the parental strand in 3′ → 5′ orientation, which is called leading strand. However, it works differently for the second strand with the 5′ → 3′ orientation, which is called lagging strand. The replication fork moves only in one direction and DNA replication goes only in the 5′ → 3′ direction, so how can this replication process be bidirectional? The paradox was solved by the discovery of Okazaki fragments (8). Instead of the continuous replication as in the leading strand, the lagging strand is produced in a discontinuous manner, which is accomplished by the synthesis of short DNA sections called Okazaki fragments. These fragments are produced from short RNA primers synthesized by an enzyme called RNA polymerase or primase. Thus, the free 3′-hydroxyl group of the RNA primer can be used by the DNA polymerase to extend the DNA. In eukaryotes, the lagging strand synthesis is carried out by the DNA polymerase a-primase complex and the primers are later removed by enzymes like ribonucleases and endonucleases. The gaps created by excised RNA are filled out by the DNA polymerase and then linked by DNA ligase to form a continuous strand of DNA.
Challenges in Delivering Gene Therapy
Yashwant Pathak in Gene Delivery, 2022
The last subfamily of retroviruses are adenoviruses, another vector used in gene therapy. Adenoviruses are non-enveloped and contain an icosahedral nucleocapsid that contains a double stranded DNA genome [11]. A unique thing about adenoviruses is that their DNA is flaked on both ends, which are called inverted terminal repeats or ITRs. This helps act as a self-primer, which promotes an enzyme called promote Primase- independent DNA synthesis and makes it important in DNA multiplication. Besides that, the ITRs help to facilitate integration of its genetic makeup into the genome of the host cell [11, 12]. Due to adenoviral vectors and their low pathogenicity and mild symptoms, these vectors can be highly favorable for gene therapy. These viruses also come in use with their ease of manipulation with recombinant DNA, as well as their ability to transduce foreign genes into proliferating cells [12–14]. The most notable use of adenoviral vectors in gene therapy was its early use in clinical trials for cystic fibrosis, which is an inherited disease that causes the cells in the lungs to produce mucus and fluids, causing severe damage to the lungs and other organs located in the general area. A subsection in adenoviral vectors is the adeno-associated virus (AAV) use as a vector. An easy explanation of AAV vectors compared to regular adenoviral vectors is that AAV is a small, slower version of adenoviral vectors. AAVs have a lower packaging capacity, lower protein levels, and take longer for genes to express while gene expression lasts longer. With a low packing size, AAVs must limit their treatment under a specific size, which is a disadvantage of using these viruses as vectors. However, depending on the situation, AAVs trigger low levels of immune response, thus allowing fewer of the vectors to be killed off by T-lymphocytes. Last, adenoviral vectors have much larger packing sizes, allowing more variability in adding a specific gene, while causing an onset of expression relatively fast at 16–24 hours [15].
Potential therapeutic targets for Mpox: the evidence to date
Published in Expert Opinion on Therapeutic Targets, 2023
Siddappa N Byrareddy, Kalicharan Sharma, Shrikesh Sachdev, Athreya S. Reddy, Arpan Acharya, Kaylee M. Klaustermeier, Christian L Lorson, Kamal Singh
In addition to DNA polymerase, there are several other enzymatic proteins which participate in poxvirus genome replication. These include helicase-primase, and topoisomerase enzymes. Both are essential for poxvirus genome replication [25]. For example, amenamevir, a helicase-primase inhibitor of variola zoster virus has been approved in Japan, and pritelivir, an inhibitor of herpesvirus helicase-primase is in phase 3 clinical trials. Since, the poxvirus helicase-primase complex is essential for viral replication, it is an important therapeutic antiviral target. Efforts have been made to develop viral topoisomerase inhibitors. For example, (+)-Rutamarin inhibits Epstein – Barr virus topoisomerase at low micromolar IC50 [136]. The reported structure of variola virus topoisomerase can also aid in the drug-discovery efforts against MPX [137].
Drugs repurposing for SARS-CoV-2: new insight of COVID-19 druggability
Published in Expert Review of Anti-infective Therapy, 2022
Sujit Kumar Debnath, Monalisha Debnath, Rohit Srivastava, Abdelwahab Omri
Two overlapping open reading frames encode four structural proteins and sixteen nonstructural proteins (NSP) (ORFs), involved in the replication/translation process of SARS-CoV-2 [37]. The structural proteins are spike glycoprotein (S), membrane (M), nucleocapsid (N), and envelope (E). Out of them, the S protein is responsible for viral entry into the host. The nonstructural proteins include papain-like proteases (NSP3), 3-chymotrypsin like protease (3-CL or Mpro/ NSP5), endonuclease (NSP15), exoribonuclease (NSP14), helicase-triphosphatase (NSP13), primase complex (NSP7-NSP8), N7 methyltransferases (NSP10), RNA-dependent RNA polymerase (NSP12), and 2’O-methyltransferase (NSP16) [55]. Apart from these, some accessory proteins also persist in their structure. Different repurposing drugs have been explored to target these proteins to stop virus propagation (Figure 2).
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.
Related Knowledge Centers
- Bacteria
- DNA
- DNA Replication
- Enzyme
- Exonuclease
- Helicase
- Rna
- Rna Polymerase
- DNA Replication
- Rna Polymerase
- Primer
- DNA
- Rna
- Primosome