scholarly journals Molecular Dissection of the Primase and Polymerase Activities of Deep-Sea Phage NrS-1 Primase-Polymerase

2021 ◽  
Vol 12 ◽  
Author(s):  
Fengtao Huang ◽  
Xueling Lu ◽  
Chunxiao Yu ◽  
Piotr Sliz ◽  
Longfei Wang ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Deep Sea ◽  
Catalytic Domain ◽  
De Novo ◽  
Dna Polymerases ◽  
Initiation Site ◽  
Polymerase Activity ◽  
Single Protein ◽  
Key Residues ◽  
Inactivating Mutation

PrimPols are a class of primases that belong to the archaeo-eukaryotic primase (AEP) superfamily but have both primase and DNA polymerase activities. Replicative polymerase from NrS-1 phage (NrSPol) is a representative of the PrimPols. In this study, we identified key residues for the catalytic activity of NrSPol and found that a loop in NrSPol functionally replaces the zinc finger motif that is commonly found in other AEP family proteins. A helix bundle domain (HBD), conserved in the AEP superfamily, was recently reported to bind to the primase recognition site and to be crucial for initiation of primer synthesis. We found that NrSPol can recognize different primase recognition sites, and that the initiation site for primer synthesis is not stringent, suggesting that the HBD conformation is flexible. More importantly, we found that although the HBD-inactivating mutation impairs the primase activity of NrSPol, it significantly enhances the DNA polymerase activity, indicating that the HBD hinders the DNA polymerase activity. The conflict between the primase activity and the DNA polymerase activity in a single protein with the same catalytic domain may be one reason for why DNA polymerases are generally unable to synthesize DNA de novo.

PrimPol (Primase/Polymerase), replicating enzyme – A mini review

2020 ◽  
Vol 2 (4) ◽  
pp. 89-92
Author(s):  
Muhammad Amir ◽  
Sabeera Afzal ◽  
Alia Ishaq
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Information ◽  
Nuclear Dna ◽  
De Novo ◽  
Dna Polymerases ◽  
Test Site ◽  
Mitochondrial Dna Replication ◽  
Dna Template ◽  
Preliminary Phase

Polymerases were revealed first in 1970s. Most important to the modest perception the enzyme responsible for nuclear DNA replication that was pol , for DNA repair pol and for mitochondrial DNA replication pol  DNA construction and renovation done by DNA polymerases, so directing both the constancy and discrepancy of genetic information. Replication of genome initiate with DNA template-dependent fusion of small primers of RNA. This preliminary phase in replication of DNA demarcated as de novo primer synthesis which is catalyzed by specified polymerases known as primases. Sixteen diverse DNA-synthesizing enzymes about human perspective are devoted to replication, reparation, mutilation lenience, and inconsistency of nuclear DNA. But in dissimilarity, merely one DNA polymerase has been called in mitochondria. It has been suggest that PrimPol is extremely acting the roles by re-priming DNA replication in mitochondria to permit an effective and appropriate way replication to be accomplished. Investigations from a numeral of test site have significantly amplified our appreciative of the role, recruitment and regulation of the enzyme during DNA replication. Though, we are simply just start to increase in value the versatile roles that play PrimPol in eukaryote.

Detection and characterization of DNA polymerase activity in Toxoplasma gondii

Parasitology ◽  
1993 ◽  
Vol 107 (2) ◽  
pp. 135-139 ◽  
Author(s):  
A. Makioka ◽  
B. Stavros ◽  
J. T. Ellis ◽  
A. M. Johnson
Keyword(s):  
Toxoplasma Gondii ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Sedimentation Coefficient ◽  
Polymerase Activity ◽  
Magnesium Ions ◽  
Dna Polymerase Β ◽  
Human Dna ◽  
Approximate Molecular Weight

SUMMARYA DNA polymerase activity has been detected and characterized in crude extracts from tachzoites of Toxoplasma gondii. The enzyme has a sedimentation coefficient of 6·4 S, corresponding to an approximate molecular weight of 150000 assuming a globular shape. Like mammalian DNA polymerase α, the DNA polymerase of T. gondii was sensitive to N-ethylmaleimide and inhibited by high ionic strength. However, the enzyme activity was not inhibited by aphidicolin which is an inhibitor of mammalian DNA polymerases α, δ and ε and also cytosine-β-D-arabinofuranoside-5′-triphosphate which is an inhibitor of α polymerase. The activity was inhibited by 2′,3′-dideoxythymidine-5′-triphosphate which is an inhibitor of mammalian DNA polymerase β and γ. Magnesium ions (Mg2+) were absolutely required for activity and its optimal concentration was 6 mM. The optimum potassium (K+) concentration was 50 mM and a higher concentration of K+ markedly inhibited the activity. Activity was optimal at pH 8. Monoclonal antibodies against human DNA polymerase did not bind to DNA polymerase of T. gondii. Thus the T. gondii enzyme differs from the human enzymes and may be a useful target for the design of toxoplasmacidal drugs.

scholarly journals Amino Acid Changes within Conserved Region III of the Herpes Simplex Virus and Human Cytomegalovirus DNA Polymerases Confer Resistance to 4-Oxo-Dihydroquinolines, a Novel Class of Herpesvirus Antiviral Agents

2003 ◽  
Vol 77 (3) ◽  
pp. 1868-1876 ◽  
Author(s):  
Darrell R. Thomsen ◽  
Nancee L. Oien ◽  
Todd A. Hopkins ◽  
Mary L. Knechtel ◽  
Roger J. Brideau ◽  
...  
Keyword(s):  
Amino Acid ◽  
Herpes Simplex ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Nucleoside Analogs ◽  
Polymerase Activity ◽  
Conserved Domain ◽  
Domain Iii ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT The 4-oxo-dihydroquinolines (PNU-182171 and PNU-183792) are nonnucleoside inhibitors of herpesvirus polymerases (R. J. Brideau et al., Antiviral Res. 54:19-28, 2002; N. L. Oien et al., Antimicrob. Agents Chemother. 46:724-730, 2002). In cell culture these compounds inhibit herpes simplex virus type 1 (HSV-1), HSV-2, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), and human herpesvirus 8 (HHV-8) replication. HSV-1 and HSV-2 mutants resistant to these drugs were isolated and the resistance mutation was mapped to the DNA polymerase gene. Drug resistance correlated with a point mutation in conserved domain III that resulted in a V823A change in the HSV-1 or the equivalent amino acid in the HSV-2 DNA polymerase. Resistance of HCMV was also found to correlate with amino acid changes in conserved domain III (V823A+V824L). V823 is conserved in the DNA polymerases of six (HSV-1, HSV-2, HCMV, VZV, Epstein-Barr virus, and HHV-8) of the eight human herpesviruses; the HHV-6 and HHV-7 polymerases contain an alanine at this amino acid. In vitro polymerase assays demonstrated that HSV-1, HSV-2, HCMV, VZV, and HHV-8 polymerases were inhibited by PNU-183792, whereas the HHV-6 polymerase was not. Changing this amino acid from valine to alanine in the HSV-1, HCMV, and HHV-8 polymerases alters the polymerase activity so that it is less sensitive to drug inhibition. In contrast, changing the equivalent amino acid in the HHV-6 polymerase from alanine to valine alters polymerase activity so that PNU-183792 inhibits this enzyme. The HSV-1, HSV-2, and HCMV drug-resistant mutants were not altered in their susceptibilities to nucleoside analogs; in fact, some of the mutants were hypersensitive to several of the drugs. These results support a mechanism where PNU-183792 inhibits herpesviruses by interacting with a binding determinant on the viral DNA polymerase that is less important for the binding of nucleoside analogs and deoxynucleoside triphosphates.

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 Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site

1991 ◽  
Vol 11 (9) ◽  
pp. 4786-4795
Author(s):  
J S Gibbs ◽  
K Weisshart ◽  
P Digard ◽  
A deBruynKops ◽  
D M Knipe ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Cell Nuclei ◽  
Dna Polymerase I ◽  
Viral Dna ◽  
Gene Encoding ◽  
Simplex Virus ◽  
Polymerase I

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.

scholarly journals DNA-metabolizing enzymes in normal human lymphoid cells. VI. Induction of DNA polymerases alpha, beta, and gamma following stimulation with phytohemagglutinin

Blood ◽  
1975 ◽  
Vol 46 (4) ◽  
pp. 509-518
Author(s):  
RJ Mayer ◽  
RG Smith ◽  
RC Gallo
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Lymphoid Cells ◽  
Blood Lymphocytes ◽  
Polymerase Beta ◽  
Normal Human ◽  
Alpha Beta ◽  
Cellular Dna

At least three distinct DNA polymerases, named alpha, beta, and gamma, have been isolated from normal mammalian cells. The function of these enzymes in regard to DNA replication and repair remains unclear. Stimulation of blood lymphocytes with the plant mitogen phytohemagglutinin (PHA), is known to increase total DNA polymerase activity. In this study, we measured the change of each of these activities as lymphocytes intered a mitotic cycle. Aliquots of a pool of normal human blood lymphocytes were incubated with PHA for 0, 24, 48, and 72 hr, respectively, and the various DNA polymerase activities quantitated at each point. No significant DNA polymerase activity was detected in unstimulated cells. Low levels of polymerase beta were found at 24 hr. The average DNA content per cell doubled between 24 and 48 hr, and during this interval all three DNA polymerases increased to easily detectable levels. By far the greatest fractional increase in activity of all three polymerases was seen between 48 and 72 hr, after the average doubling of cellular DNA. In summary, these blood lymphocytes lack significant levels of DNA polymerases; stimulation with PHA induces all three of the major DNA polymerase species. In both these respects, these cells differ from other proliferating mammalian cell systems. The possible significance of this difference is discussed.

scholarly journals DNA-metabolizing enzymes in normal human lymphoid cells. VI. Induction of DNA polymerases alpha, beta, and gamma following stimulation with phytohemagglutinin

Blood ◽  
1975 ◽  
Vol 46 (4) ◽  
pp. 509-518 ◽  
Author(s):  
RJ Mayer ◽  
RG Smith ◽  
RC Gallo
Keyword(s):  
Dna Polymerase ◽  
Mammalian Cells ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Lymphoid Cells ◽  
Blood Lymphocytes ◽  
Polymerase Beta ◽  
Normal Human ◽  
Alpha Beta ◽  
Cellular Dna

Abstract At least three distinct DNA polymerases, named alpha, beta, and gamma, have been isolated from normal mammalian cells. The function of these enzymes in regard to DNA replication and repair remains unclear. Stimulation of blood lymphocytes with the plant mitogen phytohemagglutinin (PHA), is known to increase total DNA polymerase activity. In this study, we measured the change of each of these activities as lymphocytes intered a mitotic cycle. Aliquots of a pool of normal human blood lymphocytes were incubated with PHA for 0, 24, 48, and 72 hr, respectively, and the various DNA polymerase activities quantitated at each point. No significant DNA polymerase activity was detected in unstimulated cells. Low levels of polymerase beta were found at 24 hr. The average DNA content per cell doubled between 24 and 48 hr, and during this interval all three DNA polymerases increased to easily detectable levels. By far the greatest fractional increase in activity of all three polymerases was seen between 48 and 72 hr, after the average doubling of cellular DNA. In summary, these blood lymphocytes lack significant levels of DNA polymerases; stimulation with PHA induces all three of the major DNA polymerase species. In both these respects, these cells differ from other proliferating mammalian cell systems. The possible significance of this difference is discussed.

scholarly journals Polymerization activity of an alpha-like DNA polymerase requires a conserved 3'-5' exonuclease active site.

1991 ◽  
Vol 11 (9) ◽  
pp. 4786-4795 ◽  
Author(s):  
J S Gibbs ◽  
K Weisshart ◽  
P Digard ◽  
A deBruynKops ◽  
D M Knipe ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Cell Nuclei ◽  
Dna Polymerase I ◽  
Viral Dna ◽  
Gene Encoding ◽  
Simplex Virus ◽  
Polymerase I

Most DNA polymerases are multifunctional proteins that possess both polymerizing and exonucleolytic activities. For Escherichia coli DNA polymerase I and its relatives, polymerase and exonuclease activities reside on distinct, separable domains of the same polypeptide. The catalytic subunits of the alpha-like DNA polymerase family share regions of sequence homology with the 3'-5' exonuclease active site of DNA polymerase I; in certain alpha-like DNA polymerases, these regions of homology have been shown to be important for exonuclease activity. This finding has led to the hypothesis that alpha-like DNA polymerases also contain a distinct 3'-5' exonuclease domain. We have introduced conservative substitutions into a 3'-5' exonuclease active site homology in the gene encoding herpes simplex virus DNA polymerase, an alpha-like polymerase. Two mutants were severely impaired for viral DNA replication and polymerase activity. The mutants were not detectably affected in the ability of the polymerase to interact with its accessory protein, UL42, or to colocalize in infected cell nuclei with the major viral DNA-binding protein, ICP8, suggesting that the mutation did not exert global effects on protein folding. The results raise the possibility that there is a fundamental difference between alpha-like DNA polymerases and E. coli DNA polymerase I, with less distinction between 3'-5' exonuclease and polymerase functions in alpha-like DNA polymerases.

scholarly journals Synthetic Nucleotides as Probes of DNA Polymerase Specificity

2012 ◽  
Vol 2012 ◽  
pp. 1-17 ◽  
Author(s):  
Jason M. Walsh ◽  
Penny J. Beuning
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Chemical Diversity ◽  
Dna Polymerase I ◽  
Mutagenic Potential ◽  
Natural Agents ◽  
Polymerase I ◽  
Y Family ◽  
Insight Into

The genetic code is continuously expanding with new nucleobases designed to suit specific research needs. These synthetic nucleotides are used to study DNA polymerase dynamics and specificity and may even inhibit DNA polymerase activity. The availability of an increasing chemical diversity of nucleotides allows questions of utilization by different DNA polymerases to be addressed. Much of the work in this area deals with the A family DNA polymerases, for example,Escherichia coliDNA polymerase I, which are DNA polymerases involved in replication and whose fidelity is relatively high, but more recent work includes other families of polymerases, including the Y family, whose members are known to be error prone. This paper focuses on the ability of DNA polymerases to utilize nonnatural nucleotides in DNA templates or as the incoming nucleoside triphosphates. Beyond the utility of nonnatural nucleotides as probes of DNA polymerase specificity, such entities can also provide insight into the functions of DNA polymerases when encountering DNA that is damaged by natural agents. Thus, synthetic nucleotides provide insight into how polymerases deal with nonnatural nucleotides as well as into the mutagenic potential of nonnatural nucleotides.

scholarly journals New in vitro dna polymerase activity and fidelity assay reveals age-dependent changes in Arabidopsis Thaliana

2011 ◽  
Vol 2 (1) ◽  
pp. 7 ◽  
Author(s):  
Andrey Golubov ◽  
Priti Maheshwari ◽  
Andriy Bilichak ◽  
Igor Kovalchuk
Keyword(s):  
Arabidopsis Thaliana ◽  
Dna Repair ◽  
Dna Polymerase ◽  
High Performance ◽  
Dna Polymerases ◽  
Polymerase Activity ◽  
Polymerase Fidelity ◽  
Age Dependent ◽  
Dna Chain

DNA polymerase is an enzyme that adds nucleotides to the growing DNA chain during replication and DNA repair. DNA polymerase activity and fidelity are important characteristics that reflect the ability of DNA polymerase to add nucleotides and then proofread newly synthesized DNA. We have developed a protocol allowing analysis of polymerase activity and fidelity using crude Arabidopsis thaliana plant extracts. It is based on the ability of DNA polymerases in the extract to elongate the fluorescently labelled primer annealed to a short complementary template. For analysis, fluorescently labelled products were separated on a denaturing polyacrylamide gel and visualized using a high performance blot imager. Analysis of tissue prepared from 5-, 12- and 21-day-old Arabidopsis plants showed an age-dependent decrease in polymerase activity, an increase in polymerase fidelity and a tendency to an increase in exo- (endo) nucleolytic activity.

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