Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes

Craig, Roger K.; Keir, Hamish M.

doi:10.1042/bj1450225

Cited by 37 publications

(8 citation statements)

References 26 publications

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“…We had previously observed two major sedimentation forms of DNA polymerase in crude extracts of Drosophilu embryos (7.3 S and 9.0 S) and had demonstrated that a third form (5.5 S) was a proteolysis product of the 7.3-S enzymes 1121. DNA polymerase-a from calf thymus gland, rat liver, rat spleen [23], and BHK-21/C13 cells [24] and DNA polymerase-y from mouse myeloma cells 1221 have similar sedimentation coefficients.…”

Section: Discussionmentioning

confidence: 88%

“…Multiple forms of DNA polymerase from other organisms are also resolved by DEAE-cellulose chromatography, e.g. DNA polymerase-a from calf thymus gland [24], rat liver 1231, and Xrnopus embryos [25] and DNA polymerase from Euglena [I41 and from yeast [lo]. Sedimentation of the DEAE-cellulose-purified DNA polymerases on glycerol velocity gradients showed forms of 5.5-7.3 S (enzyme I), 7.3-8.3 S (enzyme II), and 7.3 -9.0 S (enzyme 111).…”

Section: Discussionmentioning

confidence: 99%

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Three Forms of DNA Polymerase from Drosophila melanogaster Embryos

Brakel

Blumenthal

1978

European Journal of Biochemistry

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DNA polymerase was purified from Drosophila melunoguster embryos by a combination of phosphocellulose adsorption, Sepharose 6B gel filtration, and DEAE-cellulose chromatography. Three enzyme forms, designated enzymes I, 11, and 111, were separated by differential elution from DEAE-cellulose and were further purified by glycerol gradient centrifugation. Purification was monitored with two synthetic primer-templates, poly(dA) . (dT)z and poly(rA) . (dT)z. At the final step of purification, enzymes I, 11, and 111 were purified approximately 1700-fold, 2000-fold and 1000-fold, respectively, on the basis of their activities with poly(dA) . (dT)x.The DNA polymerase eluted heterogeneously as anomalously high-molecular-weight molecules from Sepharose 6B gel filtration columns. On DEAE-cellulose chromatography enzymes I and I1eluted as distinct peaks and enzyme 111 eluted heterogeneously. On glycerol velocity gradients enzyme I sedimented at 5.5 -7.3 S, enzyme I1 sedimented at 7.3 -8.3 S, and enzyme 111 sedimented at 7.3-9.0 S. All enzymes were active with both synthetic primer-templates, except the 9.0 S component of enzyme 111, which was inactive with poly(rA) . (dT)z. Non-denaturing polyacrylamide gel electrophoresis did not separate poly(dA) . (dT)x activity from poly(rA) . (dT)z activity.The DNA polymerase preferred poly(dA) . (dT)x (with MgZ+) as a primer-template, although it was also active with poly(rA) . (dT)x (withMn2+), and it preferred activated calf thymus DNA to native or heat-denatured calf thymus DNA. All three primer-template activities were inhibited by N-ethylmaleimide. Enzyme activity with activated DNA and poly(dA) . (dT)x was inhibited by K + and activity with poly(rA) . (dT)E was stimulated by K + and by spermidine. The optimum pH for enzyme activity with the synthetic primer-templates was 8.5. The DNA polymerases did not exhibit deoxyribonuclease or ATPase activities. The results of this study suggest that the forms of DNA polymerase from Drosophilu embryos have physical properties similar to those of DNA polymerase-a and enzymatic properties similar to those of all three vertebrate DNA polymerases.Prokaryotic cells [l, 21 and eukaryotic cells [3 -61 contain several DNA polymerases. The roles of DNA polymerases I, 11, and I11 of Esclzerichiu coli are being investigated by a combination of genetic and biochemical techniques [2,7]. However, the roles in vivo of the vertebrate DNA polymerases a, p, and y have not been determined. Several lines of evidence suggest that DNA polymerase-a may be the primary replication enzyme in vivo [3,5]. Proof of the functions of the eukaryotic DNA polymerases would be facilitated by

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Section: Discussionmentioning

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Three Forms of DNA Polymerase from Drosophila melanogaster Embryos

Brakel

Blumenthal

1978

European Journal of Biochemistry

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“…Heterogeneity of the enzyme activity has been observed in a variety of tissues and cells such as calf thymus [34,54,55], rat liver [34], mouse myeloma [56,57] BHK cells [58] and rabbit bone marrow [59]. This heterogeneity may be due to aggregation [26], proteolysis or possibly to association with other proteins of the replication complex and these probably account for some of the difficulties in purification.…”

Section: Eukaryote Dna Polymerasesmentioning

confidence: 99%

DNA polymerases of eukaryotes

Holmes¹,

Johnston²

1975

FEBS Letters

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“…Heterogeneity of DNA polymerase a [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] has commonly been observed during its purification from a variety of cell systems (1)(2)(3)(4)(5). This has hampered the establishment of the physical structure of DNA polymerase a and there is, as yet, no general agreement on the physical properties ofthe core enzyme (6)(7)(8)(9)(10).…”

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confidence: 99%

“…Subcellular Fractionation Procedure. The separation of the cell homogenates into the nuclear and postmicrosomal supernatant fractions was according to our published procedures (3,21,22). In this case, however, the homogenization was performed in STKMD buffer (250 mM sucrose/50 mM Tris-HCl, After' loading, the native DNA-cellulose column was washed with 10 column volumes of TDE buffer/0.…”

mentioning

confidence: 99%

Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates.

Lamothe¹,

Baril²,

Chi³

et al. 1981

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

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Heterogeneity of DNA polymerase a [DNA nucleotidyltransferase (DNA-directed), EC 2.7.7.7] has commonly been observed during its purification from a variety of cell systems (1-5). This has hampered the establishment of the physical structure of DNA polymerase a and there is, as yet, no general agreement on the physical properties ofthe core enzyme (6-10). The physiological significance, if any, of this heterogeneity is not clear at this time. Johnston and coworkers (11) showed that mild treatment of a high molecular weight form (200,000-250,000 daltons) of calf thymus DNA polymerase a with 2.5 M urea converts the enzyme to a 155,000-dalton form with the release of a 50,000-to 70,000-dalton protein(s). McKune and Holmes (12) reconstituted the high molecular weight form of polymerase a from the combination of the 155,000-dalton and the 50,000-to 70,000-dalton protein (s). They report that the 50,000-to 70,000-dalton protein(s) enhances the catalytic activity of the polymerase with synthetic polydeoxyribonucleotide templates. Villani et al. (9) have recently shown a similar conversion of DNA polymerase a from Drosophila melanogaster embryos in the presence of2.8 M urea (9). The catalytic activity of the. 148,000-dalton form of DNA polymerase a from Drosophilalembryos (9) and a 156,000-dalton form from rat liver (8) were enhanced when associated with four separable proteins of 54,000-64,000 daltons.We have previously reported the isolationwof a protein (C1) from HeLa cells thattspecifically stimulates the catalytic activity of HeLa DNA polymerase a 20-fold with DNA templateprimers that contain extended single-strand regions (13). In this paper we report the isolation and purification of three forms of DNA polymerase a from synchronized HeLa cells, one ofwhich has equal catalytic activity with activated DNA and with DNA template-primers that contain extended single-strand regions. Two proteins (Cl and C2) that are necessary for its catalytic activity with the latter template-primer are resolved from the DNA polymerase a.MATERIALS AND METHODS Materials. 3H-Labeled deoxyribonucleoside triphosphates were purchased from New England Nuclear. Unlabeled deoxyribonucleoside triphosphates and poly-and oligodeoxyribonucleotide homopolymers were from P-L

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Deoxyribonucleic acid poymerase of BHK-21/C13 cells. Heterogeneity, molecular asymmetry and subcellular distribution of the enzymes

Cited by 37 publications

References 26 publications

Three Forms of DNA Polymerase from Drosophila melanogaster Embryos

Three Forms of DNA Polymerase from Drosophila melanogaster Embryos

DNA polymerases of eukaryotes

Accessory proteins for DNA polymerase alpha activity with single-strand DNA templates.

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