Genomic Instability During Aging of Postmitotic Mammalian Cells
Alvaro Macieira-Coelho in Molecular Basis of Aging, 2017
Polymerase template activities have been used to probe for single-strand breaks or gaps in nuclear chromatin and DNA isolated at different ages.158 The rationale for studying strand breaks with DNA-dependent DNA polymerases comes from the knowledge that the initial rates of deoxynucleotide incorporation by E. coli DNA polymerase I and mammalian oc-DNA polymerase, e.g., calf thymus DNA polymerase, are directly proportional to the number of 3′-OH template primer sites, i.e., strand nicks or gaps.159 Synthesis is usually determined by autoradiography, in conjunction with radiolabeled precursors, as grains over the nucleus. Results from polymerase template activity experiments are more descriptive than those from alkaline assays in the sense that values are only indirectly related to primary lesions, and there are alternative interpretations to explain incorporation differences.65
DNA-Binding Proteins and DNA-Synthesizing Enzymes in Eukaryotes
Lubomir S. Hnilica in Chromosomal Nonhistone Proteins, 2018
Cells characteristically contain multiple forms of DNA polymerases. In prokaryotic cells, three activities are known: DNA polymerase I, II, and III.13,35 Eukaryotic cells have also at least three enzymes: DNA polymerase α, β, and γ.10–14 These DNA polymerases can easily be distinguished from one another by their size, chromatographic behavior, primer template specificities, optimal ion conditions, sensitivity to N-ethylmaleimide,10–14 and immunological comparison.14a A new type of polymerase activity called δ was recently described. This enzyme has properties similar to DNA polymerase a, but has associated exonuclease activity.36 The classification and some properties of eukaryotic DNA polymerases are listed in Table 1.
The Single-Stranded DNA Binding Protein of Bacteriophage T4
James F. Kane in Multifunctional Proteins: Catalytic/Structural and Regulatory, 2019
The central protein in the T4 replication complex is the T4 DNA polymerase, the product of gene 43. We have noted that gp32 stimulates DNA synthesis by the T4 DNA polymerase when assayed on a single-stranded DNA template.19 Conversely, the E. coli and bacteriophage T7 single-stranded DNA binding proteins do not stimulate DNA synthesis by the T4 DNA polymerase. 58,60 As we have noted, the lack of stimulation by heterologous single-stranded DNA binding proteins might be a consequence of the different geometry of the complexes between the various binding proteins and DNA. More interestingly, one can imagine that the homologous reaction involves a protein-protein contact between gp43 and gp32. Such a complex has been isolated and studied.19,59 Purified T4 DNA polymerase will co-migrate with gp32 multimers in a sucrose gradient at high gp32 concentrations. The formation of this complex is dependent on the presence of the T4 DNA polymerase; no associations are observed between gp32 and E. coli DNA polymerase I or II. This association of polymerase and gp32 has been shown to require an intact carboxyterminus of the gene 32 protein.59
Gut microbiota modulate radiotherapy-associated antitumor immune responses against hepatocellular carcinoma Via STING signaling
Published in Gut Microbes, 2022
Zongjuan Li, Yang Zhang, Weifeng Hong, Biao Wang, Yixing Chen, Ping Yang, Jian Zhou, Jia Fan, Zhaochong Zeng, Shisuo Du
Total RNA was extracted from samples, purified, and their concentrations and purity determined by nanodrop nd-1000 (NanoDrop, Wilmington, DE, USA). The integrity of RNA was detected by Bioanalyzer 2100 (Agilent, CA, USA). Concentration >50 ng/μL, Rin value > 7.0, OD260/280 > 1.8, total RNA >1 μg met the downstream test. Oligo (DT) magnetic beads (Dynabeads, oligo (DT), Thermo Fisher, USA) were used to specifically capture mRNA with poly A. Captured mRNA was fragmented (NEBNext® Magnesium RNA Fragmentation Module, USA). Fragmented RNA was reversed to synthesize cDNA. Then, E. coli DNA polymerase I (NEB, USA) and RNase H (NEB, USA) were used for two strand synthesis. These DNA and RNA were transformed into DNA double strands. At the same time, the dUTP solution (Thermo Fisher, CA, USA) was added into the ends of the double stranded DNA to fill the flat ends. Then, an A deoxynucleotide was added at both ends to connect it with the connector with T deoxynucleotides at the end. The fragment was retrieved and purified by magnetic beads. The second strand was digested using the UDG enzyme (NEB, Ma, US), after which a library with fragment size of 300 bp ± 50 bp was formed by PCR. Finally, Illumina Novaseq™ 6000 (LC Bio Technology CO., Ltd. Hangzhou, China) was used to perform pair-end sequencing according to standard operations. The sequencing mode was PE150.
Particulate matter less than 10 μm (PM10) activates cancer related genes in lung epithelial cells
Published in Inhalation Toxicology, 2020
Daeun Kang, In Beom Jung, Su Yel Lee, Se Jin Park, Sun Jung Kwon, Dong Ho Park, Ji Woong Son
After extracting the total RNA from the samples, we enriched the mRNA of the eukaryotes by using Oligo(dT) magnetic beads. Adding the fragmentation buffer, the mRNA was interrupted to form short fragments (about 200 bp). Then we synthesized the first strand cDNA by random hexamer-primer using the mRNA fragments as templates. We added buffer, dNTPs, RNase H and DNA polymerase I to synthesize the second strand. We purified the double strand cDNA with the QIAquick PCR extraction kit and washed with elution buffer for end repair and poly(A) addition. Finally, we ligated sequencing adaptors to the fragments. We purified the required fragments with agarose gel electrophoresis and enriched them with PCR amplification. The library products were ready for sequencing analysis via the Illumina HiSeq™ 4000.
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
Due to their essential function(s) during the viral life cycle, helicases are clearly attractive antiviral targets regardless of the pathogen. While some helicase-targeting small molecules inhibitors have entered clinical trials, drug development against helicases remains challenging. One of these challenges is to design specific nsp13 inhibitors that compete with the natural substrate (ATP) and bind at the ATP binding site. As with many viral functional proteins and enzymes, the ATPase function of nsp13 is common to a broad array of cellular enzymes. Therefore, not only is binding essential, but substrate specificity amongst a sea of similar substrates adds an additional layer of complexity. For example, protein phosphatase 2A (PP2A), which is involved in many cellular functions, also conducts ATP hydrolysis by binding ATP through two metal ions [91]. Hence, any inhibitor that chelates metal ions can bind PP2A-like enzymes resulting into adverse toxicity profiles. Additionally, a two metal ions mechanism originally proposed for the 3ʹ – 5ʹ exonuclease function of E. coli DNA polymerase I [92] is used by many cellular polymerases and primases. Therefore, the compounds targeting ATPase function of nsp13 through metal chelating property can potentially bind to these enzymes and interfere normal cellular function. This type of challenge is not unique to drugs targeting viral proteins/enzymes, as similar issues emerged during the discovery of anti-HIV compounds. Several nucleoside reverse transcriptase inhibitors (NRTIs) inhibit polymerase γ, a mitochondrial DNA polymerase and exert mitochondrial toxicity [93–96].