scholarly journals Purification of Drosophila DNA polymerase ζ by REV1 protein-affinity chromatography

2004 ◽  
Vol 382 (2) ◽  
pp. 535-543 ◽  
Author(s):  
Ryo TAKEUCHI ◽  
Masahiko OSHIGE ◽  
Makiyo UCHIDA ◽  
Gen ISHIKAWA ◽  
Kei-ichi TAKATA ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Biochemical Properties ◽  
Affinity Column ◽  
Protein Affinity ◽  
Pyrimidine Dimers ◽  
Multicellular Organisms ◽  
Mutation Assay

Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol ζ (DNA polymerase ζ) from Drosophila melanogaster (Dmpol ζ). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol ζ was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2′,3′-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)10:1 as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol ζ showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol ζ were not detected. Drosophila REV7 interacted with Dmpol ζ in vitro, but did not influence the DNA synthesis activity of Dmpol ζ. The present study is the first report about characterization of purified pol ζ from multicellular organisms, and the second concerning the characterization of yeast pol ζ.

Biochemical characterization of human DNA polymerase i provides clues to its biological function

2001 ◽  
Vol 29 (2) ◽  
pp. 183-187 ◽  
Author(s):  
A. Tissier ◽  
E. G. Frank ◽  
J. P. McDonald ◽  
A. Vaisman ◽  
A. R. Fernàndez deHenestrosa Henestrosa ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Short Review ◽  
Biochemical Properties ◽  
Dna Polymerase I ◽  
Polymerase I

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

scholarly journals Highly Frequent Frameshift DNA Synthesis by Human DNA Polymerase μ

2001 ◽  
Vol 21 (23) ◽  
pp. 7995-8006 ◽  
Author(s):  
Yanbin Zhang ◽  
Xiaohua Wu ◽  
Fenghua Yuan ◽  
Zhongwen Xie ◽  
Zhigang Wang
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Somatic Hypermutation ◽  
Biochemical Properties ◽  
Nonhomologous End Joining ◽  
End Joining ◽  
Synthesis Mechanism ◽  
Biochemical Analyses

ABSTRACT DNA polymerase μ (Polμ) is a newly identified member of the polymerase X family. The biological function of Polμ is not known, although it has been speculated that human Polμ may be a somatic hypermutation polymerase. To help understand the in vivo function of human Polμ, we have performed in vitro biochemical analyses of the purified polymerase. Unlike any other DNA polymerases studied thus far, human Polμ catalyzed frameshift DNA synthesis with an unprecedentedly high frequency. In the sequence contexts examined, −1 deletion occurred as the predominant DNA synthesis mechanism opposite the single-nucleotide repeat sequences AA, GG, TT, and CC in the template. Thus, the fidelity of DNA synthesis by human Polμ was largely dictated by the sequence context. Human Polμ was able to efficiently extend mismatched bases mainly by a frameshift synthesis mechanism. With the primer ends, containing up to four mismatches, examined, human Polμ effectively realigned the primer to achieve annealing with a microhomology region in the template several nucleotides downstream. As a result, human Polμ promoted microhomology search and microhomology pairing between the primer and the template strands of DNA. These results show that human Polμ is much more prone to cause frameshift mutations than base substitutions. The biochemical properties of human Polμ suggest a function in nonhomologous end joining and V(D)J recombination through its microhomology searching and pairing activities but do not support a function in somatic hypermutation.

Purification and characterization of glucose-6-phosphate dehydrogenase from Eisenia fetida and effects of some pesticides and metal ions

2020 ◽  
Vol 45 (4) ◽  
pp. 373-380
Author(s):  
Naciye Kayhan ◽  
Veysel Çomaklı ◽  
Sevki Adem ◽  
Caglar Güler
Keyword(s):  
Metal Ions ◽  
Eisenia Fetida ◽  
Biochemical Characterization ◽  
Inhibitory Effect ◽  
Biochemical Properties ◽  
Affinity Column ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

AbstractObjectivesEarthworms have a large impact on the soil ecosystem. They are quite sensitive to pollutants. Purification and biochemical characterization of glucose-6-phosphate dehydrogenases (G6PD) from the earthworm species Eisenia fetida were aimed. The determination of the toxicity potentials of some soil pollutants on G6PD activity was intended.MethodsG6PD was isolated using 2′,5′-ADP-Sepharose 4B affinity column. Enzyme purity and molecular mass were determined by SDS-PAGE. Its biochemical properties investigated. The effects of some soil pollutants on the enzyme were studied in vitro.ResultsEnzyme was purified with 28% yields and 232 fold. Optimum pH and buffer concentration, optimal and stable temperature was determined as pH: 8.5, 60 mM, 25 °C and 20 °C. Its molecular weight estimated as 36 kDa. The Ni2+, Hg2+, Pb2+, Cr2+, and Fe2+ ions with IC50 values in the range of 56 ± 06−120 ± 20 μM and the diniconazole, metalaxyl, methomyl, carboxyl, and oxamyl with IC50 values in the range of 7.6 ± 1.2−77 ± 12 μM exhibited an inhibitory effect on G6PD.ConclusionsG6PD was isolated and characterized from E. fetida. Its catalytic activity decreased with very low concentration by pesticides and metal ions. The results indicated that the inhibition of G6PD may be important in the toxicity mechanism of pollutants on this earthworm.

scholarly journals Plasticity in Neuroblastoma Cell Identity Defines a Noradrenergic-to-Mesenchymal Transition (NMT)

Cancers ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2904
Author(s):  
Margot Gautier ◽  
Cécile Thirant ◽  
Olivier Delattre ◽  
Isabelle Janoueix-Lerosey
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Neuroblastoma Cell ◽  
Neuroblastoma Cells ◽  
Biochemical Properties ◽  
Poor Overall Survival ◽  
Cell Identity ◽  
Mesenchymal Transition ◽  
Neuroblastoma Cell Lines

Neuroblastoma, a pediatric cancer of the peripheral sympathetic nervous system, is characterized by an important clinical heterogeneity, and high-risk tumors are associated with a poor overall survival. Neuroblastoma cells may present with diverse morphological and biochemical properties in vitro, and seminal observations suggested that interconversion between two phenotypes called N-type and S-type may occur. In 2017, two main studies provided novel insights into these subtypes through the characterization of the transcriptomic and epigenetic landscapes of a panel of neuroblastoma cell lines. In this review, we focus on the available data that define neuroblastoma cell identity and propose to use the term noradrenergic (NOR) and mesenchymal (MES) to refer to these identities. We also address the question of transdifferentiation between both states and suggest that the plasticity between the NOR identity and the MES identity defines a noradrenergic-to-mesenchymal transition, reminiscent of but different from the well-established epithelial-to-mesenchymal transition.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

scholarly journals Involvement of deoxyribonucleic acid polymerase β in nuclear deoxyribonucleic acid synthesis

1978 ◽  
Vol 173 (1) ◽  
pp. 309-314 ◽  
Author(s):  
T R Butt ◽  
W M Wood ◽  
E L McKay ◽  
R L P Adams
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Nuclear Dna ◽  
Dna Polymerase Beta ◽  
Cell Nuclei ◽  
High Molecular Weight Dna ◽  
Dna Polymerase Alpha ◽  
Polymerase Beta

The effects on DNA synthesis in vitro in mouse L929-cell nuclei of differential extraction of DNA polymerases alpha and beta were studied. Removal of all measurable DNA polymerase alpha and 20% of DNA polymerase beta leads to a 40% fall in the replicative DNA synthesis. Removal of 70% of DNA polymerase beta inhibits replicative synthesis by 80%. In all cases the nuclear DNA synthesis is sensitive to N-ethylmaleimide and aCTP (arabinosylcytosine triphosphate), though less so than DNA polymerase alpha. Addition of deoxyribonuclease I to the nuclear incubation leads to synthesis of high-molecular-weight DNA in a repair reaction. This occurs equally in nuclei from non-growing or S-phase cells. The former nuclei lack DNA polymerase alpha and the reaction reflects the sensitivity of DNA polymerase beta to inhibiton by N-ethylmaleimide and aCTP.

scholarly journals Characterization of human pre-elafin mutants: full antipeptidase activity is essential to preserve lung tissue integrity in experimental emphysema

2007 ◽  
Vol 405 (3) ◽  
pp. 455-463 ◽  
Author(s):  
Alain Doucet ◽  
Dominique Bouchard ◽  
Marie France Janelle ◽  
Audrey Bellemare ◽  
Stéphane Gagné ◽  
...  
Keyword(s):  
Lung Tissue ◽  
Neutrophil Elastase ◽  
Tight Binding ◽  
Biochemical Properties ◽  
Van Der Waals Interactions ◽  
Tissue Destruction ◽  
Methionine Residue ◽  
Experimental Emphysema

Pre-elafin is a tight-binding inhibitor of neutrophil elastase and myeloblastin; two enzymes thought to contribute to tissue damage in lung emphysema. Previous studies have established that pre-elafin is also an effective anti-inflammatory molecule. However, it is not clear whether both functions are linked to the antipeptidase activity of pre-elafin. As a first step toward elucidating the structure/function relationship of this protein, we describe here the construction and characterization of pre-elafin variants with attenuated antipeptidase potential. In these mutants, the P1′ methionine residue of the inhibitory loop is replaced by either a lysine (pre-elafinM25K) or a glycine (pre-elafinM25G) residue. Both mutated variants are stable and display biochemical properties undistinguishable from WT (wild-type) pre-elafin. However, compared with WT pre-elafin, their inhibitory constants are increased by one to four orders of magnitude toward neutrophil elastase, myeloblastin and pancreatic elastase, depending on the variants and enzymes tested. As suggested by molecular modelling, this attenuated inhibitory potential correlates with decreased van der Waals interactions between the variants and the enzymes S1′ subsite. In elastase-induced experimental emphysema in mice, only WT pre-elafin protected against tissue destruction, as assessed by the relative airspace enlargement measured using lung histopathological sections. Pre-elafin and both mutants prevented transient neutrophil alveolitis. However, even the modestly affected pre-elafinM25K mutant, as assayed in vitro with small synthetic substrates, was a poor inhibitor of the neutrophil elastase and myeloblastin elastolytic activity measured with insoluble elastin. We therefore conclude that full antipeptidase activity of pre-elafin is essential to protect against lung tissue lesions in this experimental model.

Oligo(A)-stimulated Tetrahymena rDNA synthesis in vitro

1981 ◽  
Vol 59 (6) ◽  
pp. 396-403 ◽  
Author(s):  
Peter R. Ganz ◽  
Gyorgy B. Kiss ◽  
Ronald E. Pearlman
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Dna Polymerase ◽  
Ribosomal Rna ◽  
Replication Proteins ◽  
General Applicability ◽  
In Vitro Synthesis ◽  
Restriction Fragments ◽  
Double Stranded Dna

The synthesis of Tetrahymena rDNA has been examined using purified DNA polymerase and partially purified preparations of homologous replication enzymes (fraction IV). DNA synthesis with purified DNA polymerase alone was less than that with fraction IV enzymes. This suggested that there were additional factors in fraction IV other than DNA polymerase which contributed to or enhanced rDNA synthesis in vitro. Neither hybridization of rDNA with Tetrahymena ribosomal RNA nor preincubation of rDNA with homologous or heterologous RNA polymerase served to stimulate in vitro synthesis by fraction IV enzymes. However, when rDNA was hybridized with oligoriboadenylate, DNA synthesis using fraction IV was stimulated approximately 4- to 4.5-fold over 150 min of incubation, relative to a similarly treated but unhybridized rDNA control. Using oligoriboadenylate-hybridized EcoR1 and HindIII restriction fragments of rDNA to localize the synthesis most of the in vitro synthesis occurred within a 2.4 × 106 Mr fragment encompassing the centre of the rDNA molecule. The approach of hybridizing a synthetic homooligoribonucleotide primer to double-stranded DNA should prove to be of general applicability in designing similar template–primers in other systems for the purpose of isolating replication proteins.

scholarly journals Characterization of the gene and mRNA of the large subunit of ribulose-1,5-bisphosphate carboxylase in pea plants.

1983 ◽  
Vol 3 (4) ◽  
pp. 587-595 ◽  
Author(s):  
K K Oishi ◽  
K K Tewari
Keyword(s):  
Immunoglobulin G ◽  
Large Subunit ◽  
Rrna Genes ◽  
Mapping Technique ◽  
Affinity Column ◽  
Bisphosphate Carboxylase ◽  
Bamhi Fragment ◽  
Dna Fragment

mRNA coding for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase was obtained by fractionating chloroplast polysomes on an affinity column, using anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Approximately 20% of the polysomal RNA specifically bound to the affinity column. LS mRNA was also isolated by fractionating chloroplast polysomal RNA on sucrose gradients. The LS mRNA fraction was identified by translation in vitro followed by immunoprecipitation with anti-ribulose-1,5-bisphosphate carboxylase immunoglobulin G. Labeled LS mRNA was hybridized to a genomic digests of pea chloroplast DNA. The LS gene was localized on a 3.55-kilobase pair BamHI fragment in SalI-SmaI DNA fragment 4. The BamHI fragment containing the LS gene was cloned, and a restriction endonuclease map was constructed. The LS gene was localized on a 1.9-kbp KpnI-EcoRI fragment. The LS gene was analyzed by electron microscopy, using the R loop mapping technique. LS mRNA was colinear with the gene, and its size was 1.35 +/- 0.2 kilobase pairs. When the LS mRNA was analyzed on methylmercury agarose gels, it comigrated with the 16S rRNA. The direction of transcription of the LS gene was in the same direction as that of the rRNA genes.

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