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Principles and Techniques for Deoxyribonucleic Acid (DNA) Manipulation
Published in Hajiya Mairo Inuwa, Ifeoma Maureen Ezeonu, Charles Oluwaseun Adetunji, Emmanuel Olufemi Ekundayo, Abubakar Gidado, Abdulrazak B. Ibrahim, Benjamin Ewa Ubi, Medical Biotechnology, Biopharmaceutics, Forensic Science and Bioinformatics, 2022
Nwadiuto (Diuto) Esiobu, Ifeoma M. Ezeonu, Francisca Nwaokorie
DNA is the chemical basis of heredity. DNA molecules serve as the repository of genetic information in all cellular forms of life and some viruses. The DNA encodes all the information required to make all of the cell’s proteins. These proteins perform the life functions of a living organism and determine their features. When a cell reproduces, it passes on its information to the daughter cells. Before a cell can reproduce, it has to make a copy of its DNA. DNA replication therefore is the process by which double-stranded DNA molecules make identical copies of themselves. This process occurs naturally during cell division in prokaryotic and eukaryotic cells, in the cytoplasm and nucleus, respectively. Irrespective of where the DNA replication occurs, however, the basic process is the same in both types of cells with some minor differences (O’Donnell et al., 2013). In both cell types, replication always starts from a site called an Origin and proceeds bidirectionally; one strand moving in the clockwise direction and the other moving counter-clockwise. However, while prokaryotic chromosomes have only one Origin, eukaryotic chromosomes are characterized by thousands of replication Origins. Not all DNA replication is bidirectional, as some bacterial plasmid DNA replicate in a unidirectional mode.
Genes and Genomics
Published in Firdos Alam Khan, Biotechnology Fundamentals, 2020
DNA replication, the basis for biological inheritance, is a fundamental process occurring in all living organisms to copy their DNA. This process is “semi-conservative” in that each strand of the original double-stranded DNA molecule serves as a template for the reproduction of the complementary strand. Hence, following DNA replication, two identical DNA molecules have been produced from a single double-stranded DNA molecule. Cellular proofreading and error-checking mechanisms ensure near perfect fidelity for DNA replication. In a cell, DNA replication begins at specific locations in the genome, called “origins.” Unwinding of DNA at the origin, and synthesis of new strands, forms a replication fork. In addition to DNA polymerase, the enzyme that synthesizes the new DNA by adding nucleotides matched to the template strand, several other proteins are associated with the fork and assist in the initiation and continuation of DNA synthesis. DNA replication can also be performed in vitro (outside a cell). DNA polymerases, isolated from cells, and artificial DNA primers are used to initiate DNA synthesis at known sequences in a template molecule. The polymerase chain reaction (PCR), a common laboratory technique, employs such artificial synthesis in a cyclic manner to amplify a specific target DNA fragment from a pool of DNA (Figure 2.12).
The Cell as an Inspiration in Biomaterial Design
Published in Heather N. Hayenga, Helim Aranda-Espinoza, Biomaterial Mechanics, 2017
Helim Aranda-Espinoza, Katrina Adlerz
DNA replication is necessary during cell division so that each daughter cell gets a complete copy of DNA from the mother cell. Each of the double strands of the DNA helix serves as a template for the enzyme DNA polymerase to create complementary strands. DNA polymerase only works in one direction along DNA from the 5′ end, the end with a phosphate, to the 3′ end, the end with a hydroxyl. For the leading strand, the DNA polymerase moves along the unwinding DNA, polymerizing a continuous strand of DNA. As the DNA polymerase moves along the lagging strand, however, it polymerizes short sequences in the 5′–3′ direction and an additional enzyme called DNA ligase pieces together these Okazaki fragments.
Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system
Published in Preparative Biochemistry & Biotechnology, 2023
Eva Agustriana, Isa Nuryana, Fina Amreta Laksmi, Kartika Sari Dewi, Hans Wijaya, Nanik Rahmani, Danu Risqi Yudiargo, Astadewi Ismadara, Moch Irfan Hadi, Awan Purnawan, Apridah Cameliawati Djohan
The large-fragment Bst DNA polymerase is the most widely used in biomedical applications and is commercially produced by industrial companies. In the diagnostic test, the enzyme has been applied for the Loop-mediated isothermal amplification (LAMP) method. In contrast with PCR-based DNA amplification, LAMP does not require a denaturation process for separating two DNA strands. The method has been developed by imitating the process of DNA replication in vivo. The physical process by heating for double-stranded DNA separation can be omitted and replaced with the enzymatic process by helicase[24]. In living organisms, a DNA helicase is functionated to disclose the double-stranded DNA into single strands, and subsequently, a DNA polymerase is involved in the synthesis and polymerization of new DNA strands during DNA replication[25]. In other words, the combination of these enzymes with a lack of 5′→3′ exonuclease activity has been included in the large fragment Bst DNA polymerase, therefore, DNA amplification can be performed under an isothermal condition in a simple way using one temperature from the scratch to the end of the reaction instead of thermocycling reaction.
Usnic acid attenuates genomic instability in Chinese hamster ovary (CHO) cells as well as chemical-induced preneoplastic lesions in rat colon
Published in Journal of Toxicology and Environmental Health, Part A, 2019
Nayane Moreira Machado, Arthur Barcelos Ribeiro, Heloiza Diniz Nicolella, Saulo Duarte Ozelin, Lucas Henrique Domingos Da Silva, Ana Paula Prado Guissone, Francisco Rinaldi-Neto, Igor Lizo Limonti Lemos, Ricardo Andrade Furtado, Wilson Roberto Cunha, Alexandre Azenha Alves De Rezende, Mário Antônio Spanó, Denise Crispim Tavares
In an attempt to contribute to the understanding of MOA underlying the antigenotoxic effect previously reported by Leandro et al. (2013), the influence of UA was investigated on genotoxicity induced by mutagens with differing MOA such as DXR, H2O2, and VP-16. DXR, an anthracycline antibiotic, is a key chemotherapeutic drug employed for cancer treatment, although its use is limited by chronic and acute adverse effects (Quiles et al. 2002). Anthracyclines such as DXR are DNA topoisomerase II inhibitors. This enzyme is involved in fundamental biological processes, including DNA replication, transcription, DNA repair, and chromatin remodeling. DXR binds to DNA topoisomerase II and stabilizes an intermediate reaction in which the DNA strands are cut and covalently linked to tyrosine residues of DNA topoisomerase II, creating a ternary DXR–DNA–DNA topoisomerase II complex that alters DNA structure and impedes its synthesis (Minotti et al. 2004). Further, the quinone present in the molecular structure of DXR may be oxidized to a semiquinone radical. Semiquinone radicals react rapidly with oxygen to generate superoxide (O2−) and H2O2 that are converted to highly reactive hydroxyl radicals, inducing DNA damage (Finn, Findley, and Kemp 2011; Injac and Strukelj 2008; Venkatesh et al. 2007).
Recombinant expression and characterization of yeast Mrc1, a DNA replication checkpoint mediator
Published in Preparative Biochemistry & Biotechnology, 2020
For the continuation of species, it is important to preserve genetic information and pass it on to future generations without errors. This process relies on a complete and reliable DNA replication process. If the DNA replication process becomes unstable or out of control, the correct delivery and expression of various types of genetic information cannot be guaranteed, resulting in various defects of the organism and various diseases including cancer, and even death. In order to cope with these risks, the organism is equipped with a corresponding DNA replication checkpoint mechanism and a repair mechanism.[1] In Saccharomyces cerevisiae (S. cerevisiae), Mec1 (serine/threonine-protein kinase (ATR) in human) is a phosphatidylinositol 3-kinase-like protein kinase (PIKK), which plays a central role in DNA replication checkpoint pathway. Ddc2 (ATR Interacting Protein (ATRIP in human)) acts as a regulatory subunit of Mec1 and forms protein complexes with Mec1.[2–4] The Mec1-Ddc2 complex is recruited to the stalled replication fork by replication protein A (RPA)-single stranded DNA (ssDNA). Ddc1, Mec3, and Rad17 are the other three proteins required for replication checkpoints, which form a trimer complex whose structure is thought to be similar to the proliferating cell nuclear antigen (PCNA).[5–10] The homolog of this trimer in human is Rad9-Hus1-Rad1 (9-1-1 complex). Current study found that the activation of Mec1/Ddc2 in S. cerevisiae is regulated by the 911 complex, and this control is dependent on cell cycle progression. Another necessary protein for replication checkpoint is Rad24 (Rad17 in human), which is similar to an RFC subunit and binds to RFCs2-5 to form a complex.[11] The Rad24-RFC complex loads the Mec3-Rad17-Ddc1 complex into the location where DNA is damaged or replication is blocked, and acts as a sensor to participate in the checkpoint response.