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 in Normal and Leukemic Human Hematopoietic Cells

Blood ◽  
1974 ◽  
Vol 44 (1) ◽  
pp. 19-32 ◽  
Author(s):  
Mary Sue Coleman ◽  
John J. Hutton ◽  
F. J. Bollum
Keyword(s):  
Molecular Weight ◽  
Dna Polymerase ◽  
Peripheral Blood ◽  
Bone Marrow Cells ◽  
Dna Polymerases ◽  
Myelogenous Leukemia ◽  
Velocity Sedimentation ◽  
Low Molecular Weight ◽  
Normal Peripheral Blood ◽  
Total Dna

Abstract DNA polymerase activities were assayed in bone marrow cells and peripheral leukocytes from normal people and patients with acute myelogenous, chronic lymphocytic, and chronic myelogenous leukemia. Extracts of subcellular components were fractionated by velocity sedimentation through sucrose density gradients and assayed using activated DNA as template. Two major DNA-dependent DNA polymerases were found in human cells with molecular weights of approximately 50,000 and 200,000 daltons, respectively. The DNA polymerase of high molecular weight is located in the soluble cytoplasmic fraction and is inhibited by N-ethylmaleimide. The low molecular weight polymerase is detected in extracts of nuclei and in the soluble fraction. It is resistant to inhibition by N-ethylmaleimide. In all cell types tested, total DNA polymerase activities were much higher in cytoplasmic than in nuclear extracts. Lymphocytes purified from normal peripheral blood had three to four times as much of both the high and low molecular weight polymerase activities per cell as purified granulocytes. Leukemic myeloblasts had 10 to 20 times as much cytoplasmic DNA polymerase activity as more mature leukocytes from normal peripheral blood. In general, immature granulopoietic cells contained higher total DNA polymerase activities than more mature granulocytes, and the major increases in polymerase activities were in the high and low molecular weight cytoplasmic enzymes rather than in the nuclear enzyme.

scholarly journals Bypass of DNA interstrand crosslinks by a Rev1–DNA polymerase ζ complex

2020 ◽  
Vol 48 (15) ◽  
pp. 8461-8473
Author(s):  
Rachel Bezalel-Buch ◽  
Young K Cheun ◽  
Upasana Roy ◽  
Orlando D Schärer ◽  
Peter M Burgers
Keyword(s):  
Catalytic Activity ◽  
Dna Polymerase ◽  
Nitrogen Mustard ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Interstrand Crosslinks ◽  
Interstrand Crosslink ◽  
Dna Interstrand Crosslinks

Abstract DNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based interstrand crosslink (ICL) with an 8-atom linker between the crosslinked bases. The Rev1–Pol ζ complex was most efficient in complete bypass synthesis, by 2–3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1–Pol ζ. Rev1–Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1–Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.

Characterisation of Nuclear and Cytoplasmic DNA Polymerases From Rat Intestinal Mucosa

1974 ◽  
Vol 52 (3) ◽  
pp. 162-169 ◽  
Author(s):  
Rozanne Poulson ◽  
J. Krasny ◽  
S. H. Zbarsky
Keyword(s):  
Molecular Weight ◽  
Intestinal Mucosa ◽  
Dna Polymerases ◽  
Deae Cellulose ◽  
Maximal Activity ◽  
Dnase I ◽  
Soluble Form ◽  
High Molecular Weight Dna ◽  
A Value ◽  
Cytoplasmic Dna

Some properties of two DNA polymerases isolated from purified nuclei of rat intestinal mucosa were compared with those of a cytoplasmic DNA polymerase from the same tissue. The molecular weight of the NaCl-soluble nuclear polymerase was estimated to be 39 000, while a value of 100 000 was determined for both the Tris-soluble nuclear and cytoplasmic enzymes. The high molecular weight DNA polymerases shared many other common characteristics. Their chromatographic properties on DEAE-cellulose and phosphocellulose were identical. In the presence of 50 mM salt or 1 mM nalidixic acid the activities of the Tris-soluble nuclear and cytoplasmic enzymes were inhibited, whereas the activity of the low molecular weight NaCl-soluble nuclear enzyme was greatly enhanced. The activities of the three DNA polymerases were inhibited by sulfhydryl blocking agents. The NaCl-soluble nuclear enzyme showed maximal activity when native DNA that had been treated briefly with DNase I was supplied as the primer–template. Extensive treatment of the native DNA with DNase I, which converted 15% of it to acid-soluble form, reduced its templating activity by more than 90%. No incorporation was detected when denatured DNA was supplied as primer–template. In contrast, both native and denatured DNA served as primer–templates for the Tris-soluble nuclear and cytoplasmic DNA polymerases, and the most efficient primer–template tested was native DNA that had been rendered 15% acid soluble by digestion with DNase I.

scholarly journals Bypass of DNA Interstrand crosslinks by a Rev1-DNA polymerase ζ complex

2020 ◽  
Author(s):  
Rachel Bezalel-Buch ◽  
Young K. Cheun ◽  
Upasana Roy ◽  
Orlando D. Schärer ◽  
Peter M. Burgers
Keyword(s):  
Catalytic Activity ◽  
Dna Polymerase ◽  
Nitrogen Mustard ◽  
Dna Polymerases ◽  
Translesion Synthesis ◽  
Interstrand Crosslinks ◽  
Interstrand Crosslink ◽  
Dna Interstrand Crosslinks

AbstractDNA polymerase ζ (Pol ζ) and Rev1 are essential for the repair of DNA interstrand crosslink (ICL) damage. We have used yeast DNA polymerases η, ζ, and Rev1 to study translesion synthesis (TLS) past a nitrogen mustard-based ICL with an 8-atom linker between the crosslinked bases. The Rev1-Pol ζ complex was most efficient in complete bypass synthesis, by 2-3 fold, compared to Pol ζ alone or Pol η. Rev1 protein, but not its catalytic activity, was required for efficient TLS. A dCMP residue was faithfully inserted across the ICL-G by Pol η, Pol ζ, and Rev1-Pol ζ. Rev1-Pol ζ, and particularly Pol ζ alone showed a tendency to stall before the ICL, whereas Pol η stalled just after insertion across the ICL. The stalling of Pol η directly past the ICL is attributed to its autoinhibitory activity, caused by elongation of the short ICL-unhooked oligonucleotide (a six-mer in our study) by Pol η providing a barrier to further elongation of the correct primer. No stalling by Rev1-Pol ζ directly past the ICL was observed, suggesting that the proposed function of Pol ζ as an extender DNA polymerase is also required for ICL repair.

Nuclear DNA polymerase activities of germinating maize embryo axes

1991 ◽  
Vol 137 (5) ◽  
pp. 581-585 ◽  
Author(s):  
Samuel Meléndez-LóPez ◽  
Jorge M. Vázquez-Ramos
Keyword(s):  
Dna Polymerase ◽  
Nuclear Dna ◽  
Embryo Axes ◽  
Maize Embryo

DNA Integrity and DNA Polymerase Activity in Deteriorated Maize Embryo Axes

1988 ◽  
Vol 133 (5) ◽  
pp. 600-604 ◽  
Author(s):  
Jorge M. Vázquez Ramos ◽  
Sergio Lopez ◽  
Edgar Vázquez ◽  
Ezequiel Murillo
Keyword(s):  
Dna Polymerase ◽  
Polymerase Activity ◽  
Dna Integrity ◽  
Embryo Axes ◽  
Maize Embryo

Purification of 7α-hydroxysteroid dehydrogenase from Escherichia coli strain 080

1990 ◽  
Vol 36 (2) ◽  
pp. 131-135 ◽  
Author(s):  
Vijay Prabha ◽  
Meenakshi Gupta ◽  
D. Seiffge ◽  
K. G. Gupta
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Key Words ◽  
Ammonium Sulfate ◽  
Gel Electrophoresis ◽  
Enzyme Preparation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Hydroxysteroid Dehydrogenase ◽  
Coli Strain

Purification studies of 7α-hydroxysteroid dehydrogenase (7α-HSDH) (EC 1.1.1.159) from Escherichia coli 080 showed that 1.59-fold purification could be achieved by heating (60 °C for 10 min) the ultracentrifuged enzyme preparation, and 6.46-fold purification was achieved by subsequent precipitation with ammonium sulfate. Further purification on Sephadex G-100 gel gave 10.1-fold purification. After pooling and concentrating the active fractions obtained from the Sephadex G-100 filtration, an 11.1-fold purification was achieved using DEAE-cellulose chromatography. The purified enzyme produced a single band on polyacrylamide gel electrophoresis and its molecular weight was determined to be 54 000. The enzyme was immunogenic and showed immunoprecipitation with homologus antisera. Key words: 7α-hydroxysteroid dehydrogenase, Escherichia coli.

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 The activity of deoxyribonucleic acid polymerase in some species of algae

1972 ◽  
Vol 129 (2) ◽  
pp. 285-290 ◽  
Author(s):  
O. Th. Schönherr ◽  
H. M. Keir
Keyword(s):  
Dna Polymerase ◽  
Green Algae ◽  
Dna Polymerases ◽  
Deae Cellulose ◽  
Chlorella Pyrenoidosa ◽  
Nuclease Activity ◽  
Polymerase Activity ◽  
Ph Optimum ◽  
Blue Green Algae ◽  
Wide Range

1. The activities of DNA polymerase preparations from the algae Euglena gracilis, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Anabaena variabilis and Anacystis nidulans were measured. The blue–green algae Anabaena and Anacystis contain a 5–20-fold higher activity of the enzyme than do the green algae. DNA polymerases from the blue–green algae show a pH optimum of 9 and prefer a relatively low Mg2+concentration (1–3mm). DNA polymerases from the green algae, however, display a pH optimum between 7.5 and 8.5 and an optimum Mg2+concentration of 8mm. With all algae, a higher polymerase activity was obtained with denatured salmon sperm DNA as template than with native DNA. All four deoxyribonucleoside 5′-triphosphates must be present for full activity of the polymerases. 2. With one exception, the deoxyribonuclease activities in the preparations, measured under conditions of the DNA polymerase assay, are low compared with corresponding preparations from Escherichia coli. Chlamydomonas extracts contain a high deoxyribonuclease activity. 3. After purification on columns of DEAE-cellulose, the polymerase activity was linear over a wide range of protein concentrations, except for Chlamydomonas preparations, where the observed deviation from linearity was probably attributable to the high nuclease activity. 4. DNA polymerases from all these algae bind strongly to DNA–cellulose; 6–40-fold purifications of the enzyme were obtained by chromatography on columns of DNA–cellulose. 5. The partially purified polymerases of Euglena and Anacystis are heat-labile but become much more heat-stable when tested in the presence of DNA.

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