scholarly journals Human PrimPol Discrimination against Dideoxynucleotides during Primer Synthesis

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1487
Author(s):  
Gustavo Carvalho ◽  
Alberto Díaz-Talavera ◽  
Patricia A. Calvo ◽  
Luis Blanco ◽  
María I. Martínez-Jiménez
Keyword(s):  
Dna Replication ◽  
De Novo ◽  
Manganese Ions ◽  
De Novo Synthesis ◽  
Replicative Stress ◽  
Mitochondrial Dna Replication ◽  
Low Efficiency ◽  
A Chain ◽  
Dna Primers ◽  
Fold Excess

PrimPol is required to re-prime DNA replication at both nucleus and mitochondria, thus facilitating fork progression during replicative stress. ddC is a chain-terminating nucleotide that has been widely used to block mitochondrial DNA replication because it is efficiently incorporated by the replicative polymerase Polγ. Here, we show that human PrimPol discriminates against dideoxynucleotides (ddNTP) when elongating a primer across 8oxoG lesions in the template, but also when starting de novo synthesis of DNA primers, and especially when selecting the 3′nucleotide of the initial dimer. PrimPol incorporates ddNTPs with a very low efficiency compared to dNTPs even in the presence of activating manganese ions, and only a 40-fold excess of ddNTP would significantly disturb PrimPol primase activity. This discrimination against ddNTPs prevents premature termination of the primers, warranting their use for elongation. The crystal structure of human PrimPol highlights Arg291 residue as responsible for the strong dNTP/ddNTP selectivity, since it interacts with the 3′-OH group of the incoming deoxynucleotide, absent in ddNTPs. Arg291, shown here to be critical for both primase and polymerase activities of human PrimPol, would contribute to the preferred binding of dNTPs versus ddNTPs at the 3′elongation site, thus avoiding synthesis of abortive primers.

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.

scholarly journals MCMBP maintains genome integrity by protecting the MCM subunits from degradation

2019 ◽  
Author(s):  
Venny Santosa ◽  
Masato T. Kanemaki
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Transcriptional Activation ◽  
Damage Accumulation ◽  
Nuclear Protein ◽  
De Novo ◽  
Genome Integrity ◽  
De Novo Synthesis ◽  
Eukaryotic Dna ◽  
High Level

AbstractThe hetero-hexameric MCM2–7 helicase plays a central role in eukaryotic DNA replication. The expression of MCM2–7 is maintained at a high level for creating dormant origins, which are important for maintaining genome integrity. However, other than transcriptional activation for the de novo synthesis, little is known about how cells maintain a high level of MCM2–7. We show that human MCMBP is a short-lived nuclear protein associating mainly with MCM5, 6, and 7. Loss of MCMBP down-regulates MCM2–7, leading to replication stress and DNA-damage accumulation. Our work demonstrates MCMBP protects the MCM subunits from degradation and suggests its chaperone-like role to achieve a high level of functional MCM2–7 using the nascent and recycled subunits.

Characterization of Monocyte-Associated Factor V

1993 ◽  
Vol 70 (02) ◽  
pp. 273-280 ◽  
Author(s):  
Janos Kappelmayer ◽  
Satya P Kunapuli ◽  
Edward G Wyshock ◽  
Robert W Colman
Keyword(s):  
Monoclonal Antibody ◽  
Light Chain ◽  
Peripheral Blood ◽  
De Novo ◽  
Factor V ◽  
Blood Monocytes ◽  
De Novo Synthesis ◽  
Hep G2 ◽  
Hep G2 Cells ◽  
Peripheral Blood Monocytes

SummaryWe demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis.Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.

Platelets Stimulate Thromboplastin Synthesis in Human Endothelial Cells

1983 ◽  
Vol 49 (02) ◽  
pp. 069-072 ◽  
Author(s):  
U L H Johnsen ◽  
T Lyberg ◽  
K S Galdal ◽  
H Prydz
Keyword(s):  
Endothelial Cells ◽  
De Novo ◽  
Umbilical Vein ◽  
Time Dependent ◽  
De Novo Synthesis ◽  
Human Endothelial Cells ◽  
Tumor Promotor ◽  
Coagulation Assay ◽  
Platelet Aggregates ◽  
Umbilical Vein Endothelial Cells

SummaryHuman umbilical vein endothelial cells in culture synthesize thromboplastin upon stimulation with phytohaemagglutinin (PHA) or the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The thromboplastin activity is further strongly enhanced in a time dependent reaction by the presence of gel-filtered platelets or platelet aggregates. This effect was demonstrable at platelet concentrations lower than those normally found in plasma, it may thus be of pathophysiological relevance. The thromboplastin activity increased with increasing number of platelets added. Cycloheximide inhibited the increase, suggesting that de novo synthesis of the protein component of thromboplastin, apoprotein III, is necessary.When care was taken to remove monocytes no thromboplastin activity and no apoprotein HI antigen could be demonstrated in suspensions of gel-filtered platelets, platelets aggregated with thrombin or homogenized platelets when studied with a coagulation assay and an antibody neutralization technique.

DE NOVO SYNTHESIS OF STEROIDS BY THE TESTES OF THE HUMAN FOETUS AT MIDGESTATION

1971 ◽  
Vol 68 (1_Supplb) ◽  
pp. S135 ◽  
Author(s):  
R. S. Mathur ◽  
N. Wiqvist ◽  
E. Diczfalusy
Keyword(s):  
De Novo ◽  
De Novo Synthesis ◽  
Human Foetus

In vivo study of the biosynthesis of long-chain fatty acids using deuterated water

1995 ◽  
Vol 269 (2) ◽  
pp. E247-E252 ◽  
Author(s):  
H. O. Ajie ◽  
M. J. Connor ◽  
W. N. Lee ◽  
S. Bassilian ◽  
E. A. Bergner ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
De Novo ◽  
Chain Elongation ◽  
Deuterated Water ◽  
De Novo Synthesis ◽  
Isotopomer Distribution ◽  
Long Chain ◽  
Mass Isotopomer

To determine the contributions of preexisting fatty acid, de novo synthesis, and chain elongation in long-chain fatty acid (LCFA) synthesis, the synthesis of LCFAs, palmitate (16:0), stearate (18:0), arachidate (20:0), behenate (22:0), and lignocerate (24:0), in the epidermis, liver, and spinal cord was determined using deuterated water and mass isotopomer distribution analysis in hairless mice and Sprague-Dawley rats. Animals were given 4% deuterated water for 5 days or 8 wk in their drinking water. Blood was withdrawn at the end of these times for the determination of deuterium enrichment, and the animals were killed to isolate the various tissues for lipid extraction for the determination of the mass isotopomer distributions. The mass isotopomer distributions in LCFA were incompatible with synthesis from a single pool of primer. The synthesis of palmitate, stearate, arachidate, behenate, and lignocerate followed the expected biochemical pathways for the synthesis of LCFAs. On average, three deuterium atoms were incorporated for every addition of an acetyl unit. The isotopomer distribution resulting from chain elongation and de novo synthesis can be described by the linear combination of two binomial distributions. The proportions of preexisting, chain elongation, and de novo-synthesized fatty acids as a percentage of the total fatty acids were determined using multiple linear regression analysis. Fractional synthesis was found to vary, depending on the tissue type and the fatty acid, from 47 to 87%. A substantial fraction (24-40%) of the newly synthesized molecules was derived from chain elongation of unlabeled (recycled) palmitate.

scholarly journals An enzymatic activation of formaldehyde for nucleotide methylation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Charles Bou-Nader ◽  
Frederick W. Stull ◽  
Ludovic Pecqueur ◽  
Philippe Simon ◽  
Vincent Guérineau ◽  
...  
Keyword(s):  
Amino Acids ◽  
Thymidylate Synthase ◽  
Active Site ◽  
De Novo ◽  
Crystallographic Structure ◽  
De Novo Synthesis ◽  
Active Site Residues ◽  
Enzymatic Activation ◽  
Enzyme Cofactors ◽  
Unique Ability

AbstractFolate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.

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