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.
Affinity Modification in Biochemistry, Biology, and Applied Sciences
Dmitri G. Knorre, Valentin V. Vlassov in Affinity Modification of Biopolymers, 1989
Therefore, RNA polymerases are able to recognize definite regions of double-stranded DNAs, usually called promoters. These regions strongly differ for RNA polymerases of different origin. Promoters used by some definite polymerase bear common features. Thus, all promoters recognized by the most-studied E. coli RNA polymerase have rather similar sequences in the region preceding the transcribed part of the template (Pribnow box) and in the region residing nearly 35 nucleotides prior to the initiation point as shown in Chapter 1, Figure 16. At the same time, DNA polymerase is unable to initiate formation of new chains and needs in a preexisting oligonucleotide (primer) complementary to some region of the template DNA. In experimental conditions this may be achieved by the use of synthetic oligonucleotides. In living systems replication starts by special RNA polymerases called primases which initiate the synthesis of short RNA primers further elongated by DNA polymerase. Primase recognizes definite points at the template DNA which thus are the points of origin of replication. At the final steps of replication short RNA pieces are eliminated by special enzymes.
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.
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.
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].
Targeting translesion synthesis (TLS) to expose replication gaps, a unique cancer vulnerability
Published in Expert Opinion on Therapeutic Targets, 2021
Sumeet Nayak, Jennifer A. Calvo, Sharon B. Cantor
Conventionally, RS deriving from DNA lesions are thought to be countered by lesion tolerance mechanisms such as translesion synthesis (TLS). Lower-fidelity TLS polymerases facilitate polymerization across from a DNA lesion in cases where the high-fidelity DNA replicative polymerases stall and cannot replicate past the lesion [31]. One critical point of regulation that engages TLS is the mono-ubiquitination of lysine 164 (K164) of proliferating cell nuclear antigen (PCNA) that is mediated by the RAD18/RAD6 ubiquitin ligases, which initiates the switch from replicative to TLS polymerases. This ubiquitination serves as a platform to recruit TLS polymerases (POL η, POL ι, POL κ, REV1, POL ζ [also known as REV3/REV7], POL θ, and POL ν) that mediate lesion bypass either in the course of DNA replication or in a process of post-replication gap filling [32–38]. By interacting with one or more TLS polymerases, PCNA functions as a ‘tool belt’ to coordinate TLS polymerases in a concerted response that initiates with a TLS polymerase such as POL η or POL κ that inserts a nucleotide at the site of the lesion [39–43]. Extension past the lesion is mediated by a distinct TLS polymerase such as POL ζ followed by a final switch to the replicative polymerases [44,45]. Alternatively, TLS can be engaged independently of PCNA ubiquitination (PCNA Ub) via a REV1 scaffold domain ‘bridge’ that interacts with several TLS polymerases [46,47]. PRIMPOL, a DNA primase and TLS polymerase, can also operate independently of PCNA to restart stalled forks or re-prime replication ahead of a lesion [48–50]. These well-orchestrated TLS polymerase switching events and their regulating mechanisms are reviewed at length in the following articles [51–54].