DNA polymerase alpha mutants from a Drosophila melanogaster cell line.

Sugino, Akio; Nakayama, Koji

doi:10.1073/pnas.77.12.7049

Cited by 42 publications

(11 citation statements)

References 26 publications

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“…Aphidicolin is a tetracyclic diterpene tetraol (Brundret et al, 1972) that inhibits DNA synthesis and the growth of eukaryotic cells. In vitro aphidicolin specifically inhibits the replicative (Bollum, 1975;Falaschi & Spadari, 1978;Weissbach, 1979) DNA polymerase a, or a-like DNA polymerase of plants (Amileni et al, 1979), with no effect on DNA polymerases #6 and y (Bucknall et al, 1973;Ikegami et al, 1978;Pedrali-Noy & Spadari, 1979;Krokan et al, 1979;Kwant & van der Vliet, 1980;Sugino & Nakayama, 1980;Sala et al, 1980Sala et al, , 1981Huberman, 1981;and references therein). Direct and unequivocal demonstration of selective inhibition of nuclear DNA replication by aphidicolin in both animal (Geuskens et al, 1981) and plant cells (Sala et al, 1981) has been achieved by electron-microscope radioautography.…”

mentioning

confidence: 99%

Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study

Hardt¹,

Pedrali-Noy²,

Focher³

et al. 1981

Biochemical Journal

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A radioautographic examination of nuclear DNA synthesis in unirradiated and u.v.-irradiated HeLa cells, in the presence and in the absence of aphidicolin, showed that aphidicolin inhibits nuclear DNA replication and has no detectable effect on DNA repair synthesis. Although the results establish that in u.v.-irradiated HeLa cells most of the DNA repair synthesis is not due to DNA polymerase alpha, they do not preclude a significant role for this enzyme in DNA repair processes.

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mentioning

confidence: 99%

Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study

Hardt¹,

Pedrali-Noy²,

Focher³

et al. 1981

Biochemical Journal

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“…The inhibition of pol a in vitro by aphidicolin correlates positively with the inhibition of DNA replication in vivo (7). An additional role for pol a in DNA repair cannot be excluded (8)(9)(10)(11).…”

mentioning

confidence: 99%

“…A DNA polymerase mutant designated aph-10 has been reported from Drosophila melanogaster (7). It contains an Nethylmaleimide-sensitive polymerase that has an elevated apparent inhibitor constant (Ki) to aphidicolin that is about 10 times higher than that of the parental enzyme, although the Km (42) and in E. coli (43).…”

mentioning

confidence: 99%

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

Liu

Chang

Trosko

et al. 1983

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

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The Chinese hamster V79 cell mutant aphr4-2, selected for its resistance to aphidicolin, a specific inhibitor of DNA polymerase a (DNA nucleotidyltransferase, EC 2.7.7.7), is characterized by slow growth, UV sensitivity, and hypersensitivity to UV-induced mutation. DNA polymerase a has been purified from mitochondria-free crude extracts of the mutant and its parental wild-type cells by sequential column chromatography on DEAE-cellulose and phosphocellulose. The major DNA polymerase activity from both cell lines was found to have characteristics of the a-type polymerase: sensitivity to 0.2 M KCI, resistance to heat denaturation (450C for 15 min), an apparent Km of 5 FM for dATP, and an ability to copy poly(dT)(rA)10 but not poly(rA)J(dT)12.The crude extracts and purified DNA polymerase a from the mutant cells are not inhibited by aphidicolin (>0.6 pAM). The apparent Km for dCTP with DNA polymerase a is 1.0 ± 0.4 pM (mean ± SD) for the mutant enzyme. The polymerase from the parental cells, similarly purified, is sensitive to aphidicolin and has an apparent K. for dCTP of 10 ± 4 FM. The spontaneous mutation rate (per cell per division), determined by fluctuation analysis at the Na+/K+-ATPase (EC 3.& 1.8) locus, is higher for mutant cells (42-73 x 10-8) than for parental cells (3-16 x 10-8). These data suggest a mechanism for aphidicolin resistance of the mutant-i.e., a decrease in the Km for dCTP. The results also indicate that an altered DNA polymerase a may be intrinsically mutagenic during normal semiconservative replicative as well as during UV-induced repair syntheses.Three generic DNA polymerases in mammalian cells have been purified, characterized, and designated as a, A, and y (1). The roles of these enzymes in cellular metabolism have been difficult to identify unambiguously in the absence of mutant polymerases. Conversely, analysis of the phenotype of cells containing mutant polymerases might help to establish the contribution of DNA polymerases to the fidelity of DNA replication and repair and to determine the involvement of each polymerase in spontaneous and induced mutagenesis.Evidence accumulated over the past two decades suggests that DNA polymerase a (pol a) is primarily associated with DNA replication. (2). DNA pol a is the major activity present in replicating cells, increases during the DNA synthetic period of the cell cycle (3), and is induced when quiescent cells are stimulated to undergo DNA replication (4). Furthermore, at the time of DNA synthesis, there is evidence for the translocation of pol a from the cytoplasm to the nucleus (5, 6).Aphidicolin, a tetracyclic diterpenoid mycotoxin, specifically inhibits DNA pol a but not polymerase ( or y. The inhibition of pol a in vitro by aphidicolin correlates positively with the inhibition of DNA replication in vivo (7). An additional role for pol a in DNA repair cannot be excluded (8-11). There is controversy, however, concerning whether or not aphidicolin also inhibits DNA repair synthesis (12-14).We have previously reported (15,16) the s...

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“…Actually, hydroxyurea has been reported to cause hypermethylation (Nyce et al 1986). Hypermethylation was also found in amplified DNA of Neurospora crassa (Bull & Wootton 1984) and in amplified ribosomal RNA genes in a rat cell line (Sugino & Nakayama 1980). It was implied that hypermethylation caused by hydroxyurea and gene amplification might be linked (Nyce et al 1986).…”

Section: Analysis Of Exogenous Npt H Activitymentioning

confidence: 83%

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

Wei

Hughes

1995

Plant Cell Tiss Organ Cult

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In previous studies, tobacco protoplasts were transformed with the bacterial gene encoding neomycin phosphotransferase II (NPT II). Transformed calluses lost neomycin phosphotransferase II activity after several subcultures. Treatment of calluses with 5-azacytidine, a demethylating agent, restored enzyme activity, suggesting that methylation of npt H sequences might be responsible for loss of NPT II activity. Studies presented here were designed to test that hypothesis. Results indicated that the effect of 5-azacytidine could not be blocked by the DNA replication inhibitor, hydroxyurea, nor by the 5-azacytidine analogue, cytidine as would be expected with a DNA demethylation mechanism. The level of NPT II mRNA was not increased by 5-azacytidine. Treatment with cycloheximide, a protein synthesis inhibitor, had no effect on 5-azacytidine-increased NPT II activity. There was no increase of NPT II protein caused by 5-azacytidine, whereas 5-azacytidine increased activity of NPT II. In contrast, the auxin 2,4-D increased both the NPT II protein and activity. Assays for malate dehydrogenase demonstrated that the effect of 5-azacytidine and hydroxyurea on NPT II was not due to an overall effect on callus metabolism. In vitro studies involving standard bacterial NPT II enzyme and crude extracts from untreated and 5-azacytidine-or hydroxyurea-treated calluses showed that the activity of NPT II added to the untreated extracts was lower than the activity of NPT II added to the extracts from calluses treated with 5-azacytidine or hydroxyurea, indicating that there was an unknown factor (or factors) in callus extracts which affected the activity of NPT II and itself was affected by 5-azacytidine and hydroxyurea treatment. These results suggested that one effect of 5-azacytidine in increasing NPT II activity was posttranslational.

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DNA polymerase alpha mutants from a Drosophila melanogaster cell line.

Cited by 42 publications

References 26 publications

Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study

Aphidicolin does not inhibit DNA repair synthesis in ultraviolet-irradiated HeLa cells. A radioautographic study

Mammalian mutator mutant with an aphidicolin-resistant DNA polymerase alpha.

5-Azacytidine increases the activity of neomycin phosphotransferase II in transgenic Nicotiana tabacum: A posttranslational mechanism may play a role

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