scholarly journals Nuclease Activity of the DNA Polymerases: An Evasion of Microscopic Reversibility Leading to Fidelity in Excess of Kinetic Control Levels

2018 ◽  
pp. 1-6
Author(s):  
Sosale Chandrasekhar
Keyword(s):  
Dna Polymerases ◽  
Genetic Material ◽  
Nuclease Activity ◽  
Kinetic Control ◽  
Microscopic Reversibility ◽  
Replication Process ◽  
Proof Reading ◽  
Mechanistic Analysis ◽  
Nucleotide Addition ◽  
Thermodynamic Effects

The exonuclease and endonuclease activities of the DNA polymerases, which extend their proof-reading capabilities and maintain high fidelity in the replication process, apparently conflict with the principle of microscopic reversibility. However, in-depth mechanistic analysis of the reactions leads to fascinating insights into the kinetic and thermodynamic effects controlling the replication of the primary genetic material. Thus, nucleotide addition and excision involve different pathways, although a violation of microscopic reversibility is avoided as the overall reaction does not reach equilibrium. Intriguingly, however, this allows the fidelity levels to exceed those expected from simple kinetic control.

Inhibition by Cyclic Guanosine 3':5'-Monophosphate of the Soluble DNA Polymerase Activity, and of Partially Purified DNA Polymerase A (DNA Polymerase I) from the Yeast Saccharomyces cerevisiae

1981 ◽  
Vol 36 (9-10) ◽  
pp. 813-819 ◽  
Author(s):  
Hans Eckstein
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nuclease Activity ◽  
Polymerase Activity ◽  
Yeast Cells ◽  
Primer Binding Site ◽  
Dna Polymerase I ◽  
Yeast Saccharomyces Cerevisiae ◽  
Main Component ◽  
Polymerase I

Abstract Dedicated to Professor Dr. Joachim Kühnau on the Occasion of His 80th Birthday cGMP, DNA Polymerase Activity, DNA Polymerase A, DNA Polymerase I, Baker's Yeast DNA polymerase activity from extracts of growing yeast cells is inhibited by cGMP. Experiments with partially purified yeast DNA polymerases show, that cGMP inhibits DNA polymerase A (DNA polymerase I from Chang), which is the main component of the soluble DNA polymerase activity in yeast extracts, by competing for the enzyme with the primer-template DNA. Since the enzyme is not only inhibited by 3',5'-cGMP, but also by 3',5'-cAMP, the 3': 5'-phosphodiester seems to be crucial for the competition between cGMP and primer. This would be inconsistent with the concept of a 3'-OH primer binding site in the enzyme. The existence of such a site in the yeast DNA polymerase A is indicated from studies with various purine nucleoside monophosphates.When various DNA polymerases are compared, inhibition by cGMP seems to be restricted to those enzymes, which are involved in DNA replication. DNA polymerases with an associated nuclease activity are not inhibited, DNA polymerase B from yeast is even activated by cGMP. Though some relations between the cGMP effect and the presumed function of the enzymes in the living cell are apparent, the biological meaning of the observations in general remains open.

scholarly journals Multiple deoxyribonucleic acid polymerases from quiescent wheat embryos. Purification and characterization of three enzymes from the soluble cytoplasm and one from purified mitochondria

1979 ◽  
Vol 181 (1) ◽  
pp. 183-191 ◽  
Author(s):  
M Castroviejo ◽  
D Tharaud ◽  
L Tarrago-Litvak ◽  
S Litvak
Keyword(s):  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Dna Polymerases ◽  
Exonuclease Activity ◽  
Purification And Characterization ◽  
Physico Chemical ◽  
Proof Reading ◽  
Wheat Embryos ◽  
Template Specificity

Three DNA polymerases (A, B and C) have been purified from the soluble cytoplasm of ungerminated embryos. Mainly on the basis of chromatographic, template-specificity and salt-inhibition evidence, we have characterized the three enzymes. Other physico-chemical and enzymic properties are described. From purified mitochondria we have purified a DNA polymerase that behaves like DNA polymerase B on chromatographic and template-specificity criteria. Only highly purified enzyme B from the soluble cytoplasm showed an exonuclease activity able to degrade 3′- or 5′-labelled polydeoxyribonucleotides, as well as a ‘proof-reading’ capacity.

scholarly journals Two essential replicative DNA polymerases exchange dynamically during DNA replication and after replication fork arrest

2018 ◽  
Author(s):  
Yilai Li ◽  
Ziyuan Chen ◽  
Lindsay A. Matthews ◽  
Lyle A. Simmons ◽  
Julie S. Biteen
Keyword(s):  
Dna Replication ◽  
Single Molecule ◽  
Replication Fork ◽  
Dna Polymerases ◽  
Super Resolution ◽  
Clamp Loader ◽  
Chromosomal Dna ◽  
Chromosomal Replication ◽  
Replication Process ◽  
Cooperative Process

AbstractThe replisome is the multi-protein complex responsible for faithful replication of chromosomal DNA. Using single-molecule super-resolution imaging, we characterized the dynamics of three replisomal proteins in liveBacillus subtiliscells: the two replicative DNA polymerases, PolC and DnaE, and a processivity clamp loader subunit, DnaX. We quantified the protein mobility and dwell times during normal replication and following both damage-independent and damage-dependent replication fork stress. With these results, we report the dynamic and cooperative process of DNA replication based on changes in the measured diffusion coefficients and dwell times. These experiments show that the replisomal proteins are all highly dynamic and that the exchange rate depends on whether DNA synthesis is active or arrested. Our results also suggest coupling between PolC and DnaX in the DNA replication process, and indicate that DnaX provides an important role in synthesis during repair. Furthermore, our results show that DnaE provides a limited contribution to chromosomal replication and repair.

COVID-19: An Update on Pathogenesis and Treatment

2020 ◽  
Vol 26 ◽  
Author(s):  
Marwa Abduallah AL Dhamen ◽  
Abrar Fouad Alhashim ◽  
Hawra Hussain Alqattan ◽  
Faheem Hyder Pottoo
Keyword(s):  
Genome Structure ◽  
Genetic Material ◽  
Severe Pneumonia ◽  
Current Treatment ◽  
Nonstructural Proteins ◽  
Replication Process ◽  
Reading Frame ◽  
The Family ◽  
Transmission Pathways

: In 2019, a new virus –SARS-COV2 emerged in china which infected many people affecting mainly the respiratory system. SARS-COV2 gets transmitted by inhalation of droplets from infected persons. Symptoms start to appear after the incubation period of the virus which ranges from 2 to 14 days. In most people, symptoms are usually mild such as fever, sore throat, cough, chest tightness and fatigue. In other people, the disease might progress into severe pneumonia leading to several fatal consequences. Treatment is usually supportive and the role of antiviral is not established yet. Home isolation for mild cases is important for the prevention of the transmission of infection. Although the rate of transmission of this virus is faster than other viruses from the family such as MERS-CoV, it has a lower fatality rate. The main difference in the genome structure of this family which make it distinguishable from other viruses is its use of (+) ssRNA as its genetic material which is comprised of 5’ cap located at one end and 3' polyadenylation tract at the other end. During infection of an exposed host cell, viral-encoded protease cleaves the polyprotein that results from translation of 5’ open reading frame (ORF) of the genome, culminating in releasing of multiple nonstructural proteins such as helicase (Hel), adenosine triphosphate (ATPase) and RNA-dependent RNA polymerase (Rep). These proteins are responsible for the replication process in addition to the syntheses of the sub genomic mRNA used as transcription templet strand. In this review article we discussed the transmission pathways, genetic sequence and current treatment approach of COVID-19.

scholarly journals DNA polymerases from Chlamydomonas reinhardii. Further characterization, action of inhibitors and associated nuclease activities

1978 ◽  
Vol 171 (1) ◽  
pp. 241-249 ◽  
Author(s):  
C A Ross ◽  
W J Harris
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nuclease Activity ◽  
Heat Sensitivity ◽  
Dna Templates ◽  
Chlamydomonas Reinhardii ◽  
Synthetic Dna ◽  
Catalytically Active ◽  
Dna Primers ◽  
Deoxyribonuclease Activity

The properties of three DNA polymerase species A, B and C, purified from Chlamydomonas reinhardii were compared. DNA polymerases A and B have Km values with respect to deoxyribonucleoside triphosphates of 19 micron and 3 micron respectively. DNA polymerase A is most active with activated DNA, but will also use native DNA and synthetic RNA and DNA templates with DNA primers. DNA polymerase B is also most active with activated DNA, but will use denatured DNA and synthetic DNA templates. It is inactive with RNA templates. DNA polymerase B is completely inactive in the presence of 100 micron-heparin, which has no effect on DNA polymerase A activity. Heparin dissociates DNA polymerase B into subunits that are still catalytically active, but which heparin inhibited. DNA polymerase B possesses deoxyribonuclease activity that is inhibited by 5 micron-heparin, suggesting that the deoxyribonuclease is an integral part of the DNA polymerase moiety. DNA polymerase A is devoid of nuclease activity. DNA polymerase C is similar to DNA polymerase B in all these properties, though it is more active with RNA primers and has greater heat-sensitivity.

scholarly journals Evolution towards increasing complexity through functional diversification in a protocell model of the RNA world

2021 ◽  
Vol 288 (1963) ◽  
Author(s):  
Suvam Roy ◽  
Supratim Sengupta
Keyword(s):  
Rna World ◽  
Genetic Material ◽  
Lipid Vesicles ◽  
Replication Process ◽  
Rolling Circle ◽  
Rolling Circle Mechanism ◽  
Key Steps ◽  
Functionally Diverse ◽  
Open Question ◽  
The Impact

The encapsulation of genetic material inside compartments together with the creation and sustenance of functionally diverse internal components are likely to have been key steps in the formation of ‘live’, replicating protocells in an RNA world. Several experiments have shown that RNA encapsulated inside lipid vesicles can lead to vesicular growth and division through physical processes alone. Replication of RNA inside such vesicles can produce a large number of RNA strands. Yet, the impact of such replication processes on the emergence of the first ribozymes inside such protocells and on the subsequent evolution of the protocell population remains an open question. In this paper, we present a model for the evolution of protocells with functionally diverse ribozymes. Distinct ribozymes can be created with small probabilities during the error-prone RNA replication process via the rolling circle mechanism. We identify the conditions that can synergistically enhance the number of different ribozymes inside a protocell and allow functionally diverse protocells containing multiple ribozymes to dominate the population. Our work demonstrates the existence of an effective pathway towards increasing complexity of protocells that might have eventually led to the origin of life in an RNA world.

scholarly journals Impact of DNA polymerase choice on assessment of bacterial communities by a Legionella genus-specific next-generation sequencing approach

2018 ◽  
Author(s):  
Rui P. A. Pereira ◽  
Jörg Peplies ◽  
Ingrid Brettar ◽  
Manfred G. Höfle
Keyword(s):  
Dna Polymerase ◽  
Bacterial Communities ◽  
Dna Polymerases ◽  
Diagnostic Tools ◽  
High Fidelity ◽  
Related Factors ◽  
Community Profiling ◽  
Proof Reading ◽  
The One ◽  
The Impact

ABSTRACTThe library preparation step is a major source of bias in NGS-based studies. Several PCR-related factors might negatively influence the application of NGS tools in environmental studies and diagnostics. Among the most understudied factors are DNA polymerases. In our study, we evaluated the effect of DNA polymerase type on the characterisation of bacterial communities, more precisely Legionella, using a genus-specific NGS approach. The assay with proof-reading high fidelity KAPA HiFi showed better amplification yield than the one with widely used non-proofreading HotStarTaq. Legionella community richness metrics were significantly overestimated with HotStarTaq. However, the choice of DNA polymerase did not significantly change the community profiling and composition. These results substantiate the use of proof-reading high fidelity DNA polymerases in NGS assays and highlight the need of considering the impact of different DNA polymerases in comparative studies and future guidelines for NGS-based diagnostic tools.

The Effect of Hydroxycamptothecin in the Activity of RNA and DNA Polymerases Prepared from Murine Hepatoma Cells

1985 ◽  
Vol 13 (01n04) ◽  
pp. 23-31 ◽  
Author(s):  
B. Xu ◽  
Y.H. Ling
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Polymerases ◽  
Hepatoma Cells ◽  
Replication Process ◽  
Nuclear Rna ◽  
Dna Template ◽  
Rna And Dna ◽  
Nuclear Rna Polymerase ◽  
Slight Inhibition ◽  
Nuclear Rna Polymerase Ii

Hydroxycamptothecin (HCPT) is an antitumor alkaloid isolated from Camptotheca acuminata indigenous to China. It could reduce the activity of nuclear RNA polymerase II and I(III) of hepatoma cells. HCPT at 25-100 μ M caused a remarkable inhibition on DNA polymerase α whilst only a slight inhibition on β. The inhibitory action on α was restored by increasing amounts of enzyme or DNA template, but unchanged by varying amounts of substrate. It is suggested that HCPT may exert a stronger inhibition on DNA replication process.

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