scholarly journals DNA polymerases from Chlamydomonas reinhardii. Purification and properties

1978 ◽  
Vol 171 (1) ◽  
pp. 231-240 ◽  
Author(s):  
C A Ross ◽  
W J Harris
Keyword(s):  
Dna Polymerase ◽  
Nuclear Dna ◽  
Dna Polymerases ◽  
Deae Cellulose ◽  
Single Species ◽  
Bivalent Cation ◽  
Chlamydomonas Reinhardii ◽  
Synchronous Cultures ◽  
Purification And Properties ◽  
Calf Thymus

Three DNA polymerase activities, A, B and C, were identified in extracts of exponentially growing synchronous cultures of Chlamydomonas reinhardii, and DNA polymerases A and B were characterized in detail. Both enzymes have the same binding affinity for DEAE-cellulose at pH 7.8, but can be distinguished from each other by their behaviour on phosphocellulose and DNA-agarose. ‘Activated’ calf thymus DNA was used as template, and the pH, K+ and bivalent-cation optima were measured. DNA polymerase A sediments at 5.3 S in glycerol gradients, with an apparent mol.wt. of 90000-100000. Polymerase B sediments between 8S and 10S in 100mM-KCl, the predominant species having an apparent mol.wt. of 200000. In 200mM-KCl, polymerase B dissociates to a single species, which sediments at 5.8S. A 3S species was found in aged preparations of both enzymes. The activity of polymerase B from cells harvested during nuclear DNA synthesis is twice that found in Chlamydomonas at other times during the cell cycle.

scholarly journals Purification and properties of DNA polymerase from Bacillus caldotenax

1992 ◽  
Vol 287 (3) ◽  
pp. 971-977 ◽  
Author(s):  
J A Burrows ◽  
C R Goward
Keyword(s):  
Enzyme Activity ◽  
Dna Polymerase ◽  
Maximum Activity ◽  
Equimolar Mixture ◽  
High Concentration ◽  
Bivalent Cation ◽  
Absolute Requirement ◽  
Purification And Properties ◽  
Calf Thymus ◽  
Km Value

A thermostable DNA polymerase was prepared from Bacillus caldotenax by using a four-step chromatography procedure. The protein exists as a monomer of M(r) 94,000, has a pI of 4.9 and has no associated 3′-5′ or 5′-3′-exonuclease activities or endonuclease activity. The temperature optimum of the enzyme was about 70 degrees C and the pH for maximum activity was about 7.5. The enzyme has an absolute requirement for a bivalent cation, and maximum activity was obtained at the unusually high concentration of 70 mM-MgCl2. Mg2+ could be replaced by MnCl2 or CoCl2, with decreased activity, at the lower optimal concentrations of 1 mM and 2.5 mM respectively. Enzyme activity was inhibited in the presence of 2′,3′-dideoxy-TTP, arabinosyl-CTP and aphidicolin. Enzyme activity was stimulated with KCl concentrations of about 100 mM, and concentrations of univalent salts above about 150 mM inhibited activity. The enzyme could use activated calf thymus DNA, poly(dA).p(dT)10 or primed single-stranded phage M13 DNA as a template and maximum activity was obtained with poly(dA).p(dT)10. The enzyme was inactive on unprimed single-stranded DNA, double-stranded DNA and polyribonucleotide template/primer. The apparent Km values for individual dNTPs, determined with the other dNTPs at saturating concentrations, were 5.7 microM (dCTP), 6.3 microM (dATP, dGTP) and 6.4 microM (dTTP). The Km value for the overall incorporation of each dNTP from an equimolar mixture of all four dNTPs was 24.7 microM. The kcat. value was about 1.05 s-1. The kcat./Km value was 0.16-0.18 M-1.s-1 for individual dNTPs and 0.04 for the incorporation of an equimolar mixture of all four dNTPs. Some of the properties of the enzyme show it may be classified as an alpha-Type DNA polymerase.

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.

Primary structure of the catalytic subunit of calf thymus DNA polymerase .delta.: sequence similarities with other DNA polymerases

Biochemistry ◽  
1991 ◽  
Vol 30 (51) ◽  
pp. 11742-11750 ◽  
Author(s):  
Jian Zhang ◽  
Dominic W. Chung ◽  
Cheng Keat Tan ◽  
Kathleen M. Downey ◽  
Earl W. Davie ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Primary Structure ◽  
Dna Polymerases ◽  
Catalytic Subunit ◽  
Calf Thymus Dna ◽  
Dna Polymerase Delta ◽  
Calf Thymus ◽  
Delta Sequence ◽  
Sequence Similarities

Preparation of an antibody against a maize DNA polymerase holoenzyme: identification of the polymerase catalytic subunit

1994 ◽  
Vol 72 (6) ◽  
pp. 818-822 ◽  
Author(s):  
P. Coello-Coutiño ◽  
E. García-Ramírez ◽  
J. M. Vázquez-Ramos
Keyword(s):  
Molecular Weight ◽  
Catalytic Activity ◽  
Molecular Mass ◽  
Key Words ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Deae Cellulose ◽  
Key Words Dna ◽  
Embryo Axes ◽  
Maize Embryo

Three different DNA polymerase activities can be separated from germinating maize axes through DEAE – cellulose chromatography. Of these, DNA polymerase 2 appears to be a replicative-type enzyme composed of several subunits. An antibody has been developed against the DNA polymerase 2 multisubunit complex, which mainly recognizes a polypeptide of molecular weight around 90 kDa. Polypeptides of molecular mass of 83, 70, 60, 55, 45, and 24 kDa are also recognized. Activity gels showed that the 90-kDa polypeptide possesses catalytic activity. DNA polymerases 1 and 3 are not recognized by the antibody and their activities are not reduced. However, DNA polymerase 2 activity is reduced by 70%. The nature of the different DNA polymerase accompanying subunits is discussed. Key words: DNA polymerases, maize embryo axes.

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 Cloning and Characterization of a Family B DNA Polymerase from the Hyperthermophilic Crenarchaeon Pyrobaculum islandicum

2000 ◽  
Vol 182 (3) ◽  
pp. 655-663 ◽  
Author(s):  
Markus Kähler ◽  
Garabed Antranikian
Keyword(s):  
Dna Polymerase ◽  
Recombinant Dna ◽  
Polyacrylamide Gel Electrophoresis ◽  
Dna Polymerases ◽  
Sodium Dodecyl ◽  
Binding Motif ◽  
Gene Encoding ◽  
Calf Thymus ◽  
Pyrobaculum Islandicum

ABSTRACT In order to extend the limited knowledge about crenarchaeal DNA polymerases, we cloned a gene encoding a family B DNA polymerase from the hyperthermophilic crenarchaeon Pyrobaculum islandicum. The enzyme shared highest sequence identities with a group of phylogenetically related DNA polymerases, designated B3 DNA polymerases, from members of the kingdom Crenarchaeota,Pyrodictium occultum and Aeropyrum pernix, and several members of the kingdom Euryarchaeota. Six highly conserved regions as well as a DNA-binding motif, indicative of family B DNA polymerases, were identified within the sequence. Furthermore, three highly conserved 3′-5′ exonuclease motifs were also found. The gene was expressed in Escherichia coli, and the DNA polymerase was purified to homogeneity by heat treatment and affinity chromatography. Activity staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed an active polypeptide of approximately 90 kDa. For the recombinant DNA polymerase from P. islandicum, activated calf thymus DNA was used as a substrate rather than primed single-stranded DNA. The enzyme was strongly inhibited by monovalent cations andN-ethylmaleimide; it is moderately sensitive to aphidicolin and dideoxyribonucleoside triphosphates. The half-life of the enzyme at 100 and 90°C was 35 min and >5 h, respectively. Interestingly, the pH of the assay buffer had a significant influence on the 3′-5′ exonuclease activity of the recombinant enzyme. Under suitable assay conditions for PCR, the enzyme was able to amplify λ DNA fragments of up to 1,500 bp.

scholarly journals Inhibition of deoxyribonucleic acid polymerases from human cells and from simian sarcoma virus by pyran

1978 ◽  
Vol 175 (2) ◽  
pp. 519-524 ◽  
Author(s):  
R A Dicioccio ◽  
B I S Srivastava
Keyword(s):  
Dna Polymerase ◽  
Lymphoblastic Leukemia ◽  
Dna Polymerases ◽  
Human Cell Line ◽  
Bivalent Cation ◽  
Molecular Weights ◽  
Bivalent Cations ◽  
Sarcoma Virus ◽  
Simian Sarcoma Virus ◽  
Alpha Beta

Pyrans are co-polymers of divinyl ether and maleic anhydride. Four pyrans of various molecular weights more potently inhibited terminal deoxyribonucleotidyltransferase (EC 2.7.7.31) from a human cell line of acute lymphoblastic leukemia origin (Molt-4) than they did DNA polymerases alpha, beta and gamma from these cells and DNA polymerase from simian sarcoma virus. For example, the concentrations of one pyran required for 50% inhibition of terminal deoxynucleotidyltransferase, DNA polymerases alpha, beta and gamma and viral DNA polymerase were 0.9, 110, 125, 35 and 47 microgram/ml respectively. Quantitatively similar results were obtained with the other pyrans. Inhibition of these enzymes by pyran was dependent on the concentrations of both the bivalent cation and template/primer or initiator in assay mixtures, but not on the concentrations of the substrate (deoxyribonucleoside 5′-triphosphate), enzyme, or bovine serum albumin. These results suggested that pyran inhibited these enzymes by complexing bivalent cations, which caused a decreased affinity of template/primer or initiator for each enzyme and a decrease in enzyme activity.

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