HeLa DNA polymerase ∝ activity in vitro: specific stimulation by a non-enzymic protein factor

Novak, Bill; Baril, Earl F.

doi:10.1093/nar/5.1.221

Cited by 52 publications

(35 citation statements)

References 35 publications

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“…Forms ofpol a have also been described that may use accessory factors to augment enzymatic capabilities (24,25). pol y has been found to be the principal or sole DNA polymerase in mitochondria (26)(27)(28); however, most of the pol y activity is normally found in the cytosol (exclusive of mitochondria) and nuclear fractions (22,26), and the function(s) of pol y in uninfected cells remains unknown.…”

Section: Discussionmentioning

confidence: 99%

Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma.

Kollek¹,

Goulian²

1981

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

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The initial event in the replication cycle of parvovirus H-1 is conversion of the single-stranded linear viral DNA to the double-stranded linear replicative form. We describe here detection of an activity in uninfected cell extracts that carries out this reaction. The activity was purified and identified as DNA polymerase y.H-1 is an autonomous (helper-independent) member ofthe parvovirus group (1, 2). Its genome consists of a linear singlestranded (ss) DNA (==5 kilobases) with stable hairpin structures at both ends. After attachment to a susceptible cell and uptake of viral DNA, infecting viral ss DNA must first be converted to the double-stranded (ds) replicative form (RF) by synthesis of the complementary strand. The RF undergoes replication, probably by a continuous (as opposed to semidiscontinuous) 5' --3' displacement mechanism on both strands (1, 3-5), followed by synthesis and packaging of viral ss DNA. Given the small size of the genome and the known coding requirements for viral capsid polypeptides, it is assumed that replication must depend almost entirely on host functions, as is the case for several other prokaryotic and eukaryotic small DNA viruses.We are studying parvoviral DNA replication as a model for eukaryotic DNA replication and describe here studies on the enzymatic requirements for synthesis of parental RF from infecting viral ss DNA. Relatively little is known from in vivo experiments about this initial step in parvoviral DNA replication. The 3'-terminal foldback could provide a base-paired priming point for synthesis of the complementary strand (1, 2, 6-10), and it is assumed that this functions as the initiation site for synthesis of parental RF in vivo. Although evidence for the viral DNA to RF reaction in vivo has been reported (11,12), this step has been generally difficult to study, in part due to high particle/ infectivity ratios.In (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) mCi/Amol). In an alternative procedure, assay 4b, ATP, and phosphoenolpyruvate were replaced by 40 mM NaOAc and MgCl2 was reduced from 10 mM to 7 mM. All assay mixtures also contained 1 mM dithiothreitol and bovine plasma albumin (0.2 mg/ml).Results for response to inhibitors represent % activity remaining in the presence ofinhibitor compared with 100% in the absence of inhibitor. The concentration of 2',3'-dideoxythymidine-5'-triphosphate (ddTTP) was the same as that of dTJP; aphidicolin (gift of A. H. Todd, Imperial Chemical Industries, London) was at 3 Ag/ml; N-ethylmaleimide (MalNEt) was at 6 mM. All assays were carried out in a total volume of 30 ILI, at 370C, for 30 min (assays 1, 2, and 3) or 60 min (assay 4) (unless stated otherwise). DNA synthesis was determined by incorporation of radioactivity from dNTP into acid-insoluble material. One unit of polymerase activity = 1 nmol of total dNMP incorporated in 60 min at 37C.Fractionation of NB Cell Extracts. Cytosol was prepared from uninfected NB cells by disruption with a Dounce homogenizer followed by centrifugation to remove nuclei.Whole cell...

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Section: Discussionmentioning

confidence: 99%

Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma.

Kollek¹,

Goulian²

1981

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

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“…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.…”

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confidence: 98%

“…Form I [3H]DNA of simian virus 40 (SV40) was a gift from H. Ozer (Hunter College). 3H-Labeled Escherichia coli DNA was prepared as described (13). Native and denatured DNA-cellulose were prepared according to the procedure of Alberts and Herrick (20).…”

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confidence: 99%

“…Native and denatured DNA-cellulose were prepared according to the procedure of Alberts and Herrick (20). All other reagents were of reagent or ultra-pure grade and have been described elsewhere (13,15,21).…”

mentioning

confidence: 99%

“…HeLa S3 cells were grown according to our published procedures (13,21) in Joklik's modified Eagle's medium supplemented with 3.5% each of irradiated calf and fetal bovine serum (Irvine Scientific, Irvine, CA). Cells were synchronized by the double hydroxyurea block technique and judged free from Mycoplasnw contamination by our previously described procedure (13,21). Subcellular Fractionation Procedure.…”

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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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A mammalian DNA polymerase alpha holoenzyme functioning on defined in vivo-like templates.

Hübscher¹,

Gerschwiler²,

McMaster³

1982

The EMBO Journal

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In analogy to the Escherichia coli replicative DNA polymerase III we define two forms of DNA polymerase alpha: the core enzyme and the holoenzyme. The core enzyme is not able to elongate efficiently primed single‐stranded DNA templates, in contrast to the holoenzyme which functions well on in vivo‐like template. Using these criteria, we have identified and partially purified DNA polymerase alpha holoenzyme from calf thymus and have compared it to the corresponding homogeneous DNA polymerase alpha (defined as the core enzyme) from the same tissue. The holoenzyme is able to use single‐stranded parvoviral DNA and M13 DNA with a single RNA primer as template. The core enzyme, on the other hand, although active on DNAs treated with deoxyribonuclease to create random gaps, is unable to act on these two long, single‐stranded DNAs. E. coli DNA polymerase III holoenzyme also copies the two in vivo‐like templates, while the core enzyme is virtually inactive. The homologous single‐stranded DNA‐binding proteins from calf thymus and from E. coli stimulate the respective holoenzymes and inhibit the core enzymes. These results suggest a cooperation between a DNA polymerase holoenzyme and its homologous single‐stranded DNA‐binding protein. The prokaryotic and the mammalian holoenzyme behave similarly in several chromatographic systems.

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HeLa DNA polymerase ∝ activity in vitro: specific stimulation by a non-enzymic protein factor

Cited by 52 publications

References 35 publications

Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma.

Synthesis of parvovirus H-1 replicative form from viral DNA by DNA polymerase gamma.

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

A mammalian DNA polymerase alpha holoenzyme functioning on defined in vivo-like templates.

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