Studies on the Purification and Properties of a 6.8‐S DNA Polymerase Activity Found in Calf‐Thymus DNA Polymerase‐α Fraction

Hesslewood, Ian P.; Holmes, Andrew M.; Wakeling, W. F.; Johnston, Irving R.

doi:10.1111/j.1432-1033.1978.tb12148.x

Cited by 50 publications

(16 citation statements)

References 29 publications

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“…Recent reports on the purification of DNA polymerase a do not yet give unequivocal data on the molecular weight of the catalytically active component of the enzyme (6,(20)(21)(22). Highly purified preparations of DNA polymerase a from various animal sources still contain several different components with Mrs between 50,000 and 150,000 (6,(20)(21)(22).…”

mentioning

confidence: 99%

Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha.

Grummt¹,

Waltl²,

Jantzen³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

138

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By equilibrium dialysis a diadenosine 5',5"'-P',P2-tetraphosphate (Ap4A) This enzyme was applied on a 15 X 1.5 cm column of native calf thymus DNA-cellulose. The column was washed with' 200 ml of 20 mM Tris1HCI, pH 8.0/7 mM 2-mercaptoethanol/20% glycerol and the enzyme was'eluted with a 500-ml salt gradient (0-200 mM potassium chloride). The' polymerase a-containing fractions were concentrated by vacuum dialysis and further purified by gel filtration. The enzyme solution (4 ml) was applied qn a Bjo-Gel A 0.5 column Abbreviation: Ap4A, diadenosine 5',5"-PlP4-tetraphosphate.

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mentioning

confidence: 99%

Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha.

Grummt¹,

Waltl²,

Jantzen³

et al. 1979

Proc. Natl. Acad. Sci. U.S.A.

138

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“…However, studies with two highly purified species of DNA polymerase a from mouse myeloma cells have indicated the presence of two different subunits that failed to show any sequence homology on tryptic peptide maps (8). In contrast, Hesslewood et al (6) have shown that one form of rat liver DNA polymerase a activity could be converted to a second form by exposure to mild urea treatment. Recently, we have shown the presence of three forms of DNA polymerase a activity in cultured human neuroblastoma IMR-32 cells (12,13).…”

mentioning

confidence: 87%

“…The existence of multiple forms of DNA polymerase a activity in normal and tumor cells has been well established (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), but it is not yet clear whether these multiple forms arise from a single native protein. However, studies with two highly purified species of DNA polymerase a from mouse myeloma cells have indicated the presence of two different subunits that failed to show any sequence homology on tryptic peptide maps (8).…”

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confidence: 99%

Probable involvement of a glycoconjugate in IMR-32 DNA synthesis: decrease of DNA polymerase alpha 2 activity after tunicamycin treatment.

Bhattacharya¹,

Basu²

1982

Proc. Natl. Acad. Sci. U.S.A.

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Multiple forms of DNA polymerase a activity (al, a2, and a3) from human neuroblastoma IMR-32 cells untreated or treated with tunicamycin (3 ,ug/ml) were separated by DEAE-cellulose column chromatography. Loss of40-60% ofDNA polymerase a2 activity was observed in tunicamycin-treated cells. Ricin IB, a subunit of intact ricin (Mr, 64,000), was found to be a specific inhibitor of DNA polymerase a2 isolated from control IMR-32 cells. However We report here that tunicamycin (19), which blocks the synthesis of N-acetylglucosaminylpyrophosphorylpolyisoprenol and thereby inhibits the glycosylation of protein (20-23), specifically decreases the activity of DNA polymerase a2.EXPERIMENTAL PROCEDURE Nucleotides and Polynucleotides. 3H-Labeled deoxynucleotides were purchased from New England Nuclear and ICN. Synthetic polynucleotide templates and oligonucleotide primers were obtained from either Miles Laboratories or P-L Biochemicals. Template-primer hybridization was performed according to Wickremasinghe (24). A mixture of template and primer (20:1) in 10 mM NaCl was heated for 10 min at 550C, cooled at room temperature, and stored at -20'C. In the text that follows, subscripts denote chain lengths of polynucleotide primers. Tunicamycin (lot NCI-2) was a gift from John D. Douros (National Cancer Institute).Cell Culture. Cultures of human neuroblastoma IMR-32 cells (a gift from S. Brooks) were grown in 250-ml (75-cm2) Falcon plastic flasks containing 15-20 ml of minimal essential medium supplemented with 10% fetal calf serum (GIBCO) (14). The medium also contained 100 units of penicillin/streptomycin (GIBCO) per ml to prevent microbial growth. Incubation was carried out in a humidified atmosphere of 97.5% air/2.5% CO2. The medium was changed twice weekly, and cells were subcultured upon reaching confluence. The experimental cells were maintained in minimal essential medium containing tunicamycin (3 ,ug/ml) for 40 hr to obtain maximal inhibition of DNA synthesis. The cells were harvested with 5.0 ml of phosphate-buffered saline (pH 7.2) containing 0.1% EDTA per T flask. After pelleting by centrifugation, the cells were suspended in 2-3 vol of 0.32 M sucrose and stored at -200C.Separation of DNA Polymerases on Sucrose Density Gradients. Sucrose density gradients were prepared by using 5-20% sucrose in 10 mM Tris HCl, pH 8.0/100 mM KCVl1 mM mercaptoethanol as described (14). Aliquots (0.3-0.4 ml) ofsonicated IMR-32 supernatant were layered on the gradients (4.5 ml) and centrifuged for 16 hr at 149,000 X g in a Beckman SW 50.1 rotor. Fractions (0.25 ml) were collected from the bottom with a peristaltic pump. DNA polymerase a and f8 activities were found in fractions 5 and 14, respectively.Isolation of Multiple Forms of DNA Polymerase a by DEAE-Cellulose Column Chromatography. The soluble supernatants from tunicamycin-treated or control samples were loaded on separate, identical DEAE-cellulose columns (2 X 14 cm) that had been equilibrated with 100 ml of 50 mM potassium phosphate, pH 7.6/1 mM mercaptoethanol. The proteins...

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“…wt. polypeptides (50 000 -70 000), in calf thymus (Hesslewood et al, 1978), in rat liver (Mechali et al, 1980) and in Drosophila melanogaster (Villani et al, 1980). Others have reported that the DNA polymerase ca, purified from various sources, consists of several polypeptides of low mol.…”

Section: Introductionmentioning

confidence: 99%

Active polypeptide fragments common to prokaryotic, eukaryotic, and mitochondrial DNA polymerases.

Scovassi¹,

Torsello²,

Plevani³

et al. 1982

The EMBO Journal

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( ac-tivity is detectable and the pattern of the high mol. wt. cluster is similar to that observed in E. coli extracts (which also lack low mol. wt. peptides). Also in mitochondria from higher and lower eukaryotes only high mol. wt. species are observed, and the active band(s) range from 70 000 to 145 000 daltons. Our results indicate that the stmcture of DNA polymerase has been highly conserved during evolution, so that an active fragment of mol. wt. 270 000 is ahvays found in prokaryotic enzymes and in the replicative species of eukaryotic and mitochondrial DNA polymerases; at a certain stage in evolution, another species of low mol. wt. DNA polymerase (3 or (-like) appears.

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Studies on the Purification and Properties of a 6.8‐S DNA Polymerase Activity Found in Calf‐Thymus DNA Polymerase‐α Fraction

Cited by 50 publications

References 29 publications

Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha.

Diadenosine 5',5'''-P1,P4-tetraphosphate, a ligand of the 57-kilodalton subunit of DNA polymerase alpha.

Probable involvement of a glycoconjugate in IMR-32 DNA synthesis: decrease of DNA polymerase alpha 2 activity after tunicamycin treatment.

Active polypeptide fragments common to prokaryotic, eukaryotic, and mitochondrial DNA polymerases.

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