Isolation of DNA polymerase gamma from an adenovirus 2 DNA replication complex

Ito, Kazuhiko; Arens, Max Q.; Green, M

doi:10.1128/jvi.15.6.1507-1510.1975

Cited by 40 publications

(10 citation statements)

References 18 publications

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“…We have reinvestigated the question of DNA polymerase involvement in adenovirus DNA synthesis, both by analysis of the polymerase present in the "replication complex" and by direct inhibition of DNA synthesis in the complex by phosphonoacetic acid. In contrast to Ito et al (3,4), our findings tend to support a role for DNA polymerase a in adenovirus DNA synthesis.…”

contrasting

confidence: 99%

Adenovirus DNA synthesis in vitro in an isolated complex

Frenkel

1978

J Virol

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DNA-protein complexes isolated from adenovirus-infected cells by a modification of the M-band technique were used as an in vitro system for the study of adenovirus DNA replication. The synthesis in vitro was semiconservative, inhibited by N-ethylmaleimide, and stimulated by ATP. Studies on DNA-negative mutants of adenovirus showed that the DNA synthesis in vitro represents a continuation of adenovirus DNA replication in vivo. DNA synthesis in vitro was inhibited 38% by 20 microgram of phosphonoacetic acid per ml, which is several-fold higher than the inhibition obtained with purified DNA polymerase beta or gamma, but was similar to the degree of inhibition of DNA polymerase alpha. DNA synthesis in complexes from uninfected cells was much less sensitive to inhibition by phosphonoacetic acid. In addition, complexes from infected cells contained a greater proportion of the alpha-polymerase than complexes from uninfected cells, suggesting that an association of alpha-polymerase with the replication complex may be occurring during adenovirus infection, with subsequent utilization of the alpha-polymerase for viral DNA synthesis.

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confidence: 99%

Adenovirus DNA synthesis in vitro in an isolated complex

Frenkel

1978

J Virol

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“…The DNA polymerase obtained by two fractionation procedures following rupture of the sperm nucleus appears to be the enzyme present in the DNA-synthesizing complex (Witkin et al, 1975;Witkin & Bendich, 1977;Witkin et al, 1977a). DNA polymerase has been found associated with other endogenous DNA-synthesizing components from human lymphoid cells (Yoshida & CavaUeri, 1977), rat brain (Witkin & Schumaker, 1977) and adenovirus-infected cells (Ito, Arens & Green, 1975;Brison, Kendinger & Wilhelm, 1977) and it may therefore be involved in specific DNA synthesis. The retention of this enzyme by the highly specialized male gametes, which have lost cytoplasm and achieved a condensed nucleus during spermatogenesis, suggests that the enzyme might have a function after fertilization.…”

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confidence: 99%

Properties of a DNA polymerase from purified nuclei and DNA-synthesizing complexes of human spermatozoa

Ss¹

1980

Reproduction

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A DNA polymerase was isolated from human spermatozoa. In one procedure, spermatozoa were decapitated with detergent, the heads purified and then lysed with dithiothreitol, trypsin and deoxyribonuclease. DNA polymerase was isolated from the lysate by sedimentation through an 18% Metrizamide solution, solubilization with 0.8 M-KCl-0.5% Triton X-100 and sequential chromatography on DEAE cellulose, phosphocellulose and hydroxylapatite. Alternatively, the heads of intact spermatozoa, untreated with detergent, were lysed as above; the subsequent Metrizamide pellet fraction was isolated and further fractionated by gel filtration and buoyant density centrifugation. The enzyme in this fraction was solubilized with KCl-Triton X-100. Characterization by velocity centrifugation and phosphocellulose chromatography revealed that it possessed properties indistinguishable from those of the enzyme purified from isolated sperm nuclei. The DNA polymerase had an apparent molecular weight of 79,000-89,000, Mn2+ (1 mM) was the preferred divalent cation and ativity was inhibited by concentrations of potassium phosphate greater than 10 mM. The synthetic template preferences of the enzyme were dT12-18 . poly rA > poly(dA-dT) > dT12-18 . poly dA; no activity was observed with dG12-18 . poly rC or dT10.

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“…Equal portions were counted, and the fractions corresponding to the interpeak region (as indicated in Fig. 3A) were pooled (105 ml) and adjusted to 0.5% Triton X-100 and 0.4 M ammonium sulfate (13). After 1 h at 00C with constant stirring, the mixture was passed at a flow rate of 300 pl/min through a DEAE-cellulose column (2.8 by 1.5 cm) equilibrated with a buffer containing 20 mM Trishydrochloride (pH 7.9), 1 mM dithiothreitol, 0.4 M ammonium sulfate, and 0.5% Triton X-100.…”

Section: (See Below)mentioning

confidence: 99%

“…After incubation, both the products and the template in this fraction consisted of short fragments (6 to 9S and 18S, respectively) of DNA. The main DNA polymerase activity involved in the membrane-associated replication system has been recently identified as belonging to the y class (13,14).…”

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Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei

Brison¹,

Kédinger²,

Wilhelm³

1977

J Virol

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When HeLa cell nuclei, isolated 17 h after infection with adenovirus type 2 (Ad2), were extracted with 200 mM ammonium sulfate, Ad2 nucleoprotein complexes were selectively released. These complexes contained a DNA polymerase activity that corresponded to DNA polymerase molecules actively engaged in Ad2 DNA replication. Under our high-salt (200 mM ammonium sulfate) incubation conditions, where no reinitiation occurred, full-length Ad2 DNA chains were synthesized by elongation of chains that had been initiated in vivo. This conclusion was further supported by density labeling experiments indicating that the in vitro DNA synthesis was semiconservative. Evidence is presented suggesting that at least part of the DNA polymerase molecules engaged in Ad2 DNA replication belong to the gamma class.

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Isolation of DNA polymerase gamma from an adenovirus 2 DNA replication complex

Cited by 40 publications

References 18 publications

Adenovirus DNA synthesis in vitro in an isolated complex

Adenovirus DNA synthesis in vitro in an isolated complex

Properties of a DNA polymerase from purified nuclei and DNA-synthesizing complexes of human spermatozoa

Enzymatic properties of viral replication complexes isolated from adenovirus type 2-infected HeLa cell nuclei

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