Induction of DNA polymerase in mouse L cells

Chang, Lucy M.S.; Brown, Marie Scott; Bollum, F. J.

doi:10.1016/0022-2836(73)90349-5

Cited by 219 publications

(56 citation statements)

References 8 publications

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“…The finding that at least the initial phase of increased polymerase activity was sensitive to the presence of NEM would suggest that either DNA polymerase-a or DNA-polymerase-y or both are involved in this response, but not DNA polymerase-po Our finding is in apparent contradiction to much recent work which has suggested that DNA polymerase-p is the major repair enzyme whilst DNA polymerase-a and DNA polymerase-yare involved in semiconservative DNA synthesis (see Chang et al 1973;Bertazzoni et al 1976;Robison and Fansler 1978;Hiibscher et al 1979). However, some authors, for example Hiibscher et al (1979), have not ruled out the possibility of some minor involvement of DNA polymerase-a and perhaps DNA polymerase-y in repair.…”

Section: Discussioncontrasting

confidence: 96%

X-ray Induced Activity of N-Ethylmaleimide-Sensitive DNA Polymerase in Chinese Hamster Cells

Ramsay¹,

Westerman²

1980

Aust. Jnl. Of Bio. Sci.

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Chinese hamster DDN cells have been exposed to low doses of X-irradiation « 15 Gy). These doses have been shown to induce significant levels of repair replication. Levels of activity of DNA polymerase were subsequently measured in cells exposed in G 1 phase of mitosis to 7·5 Gy of X-rays. Following exposure, the activity of this enzyme was observed to rise to a peak within 15 min postirradiation, then decline and then rise again over a long period. The initial rise at least was found to be sensitive to inhibition by N-ethylmaleimide, a known inhibitor of DNA polymerase-o< and DNA polymerase-y at the concentration used. It is therefore suggested that repair of X-ray-induced damage in these DON cells was at least in part dependent upon induced DNA polymerase activity and this may involve DNA polymerase-o< or DNA polymerase-y, or both.

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Section: Discussioncontrasting

confidence: 96%

X-ray Induced Activity of N-Ethylmaleimide-Sensitive DNA Polymerase in Chinese Hamster Cells

Ramsay¹,

Westerman²

1980

Aust. Jnl. Of Bio. Sci.

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“…If the 8-S enzyme were a dimer, then the 1 0 s and the 12.3-S enzymes could probably be the trimer and the tetramer of a monomeric protein with a molecular weight of about 90000. Taking into account the fact that in tissues of higher organisms the concentration of the 6 -8-S DNA polymerase is very high during active growth [3,4] it could be assumed that this might be also the case in yeast. The 3.4-S polymerase might therefore be hardly detectable in the presence of an excess of the high-molecular-weight species.…”

Section: Resultsmentioning

confidence: 99%

Absence of a Low‐Molecular‐Weight DNA Polymerase from Nuclei of the Yeast, Saccharomyces cerevisiae

Wintersberger

1974

European Journal of Biochemistry

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The DNA polymerase activity associated with purified nuclei of the yeast, Succharomyces cerevisiae, was analyzed by sucrose gradient centrifugation. Using different salt concentrations there was no polymerase activity found sedimenting in the 3 -4-S region. On the contrary, the nuclear enzyme shows a main peak around 8 S, similar to the DNA polymerases from yeast cytoplasm. Other properties tested ( e g . behaviour on ion-exchange columns, dependence of activity on salt concentration, inhibition by p-chloromercuribenzoate etc.) were also found identical with those of the cytoplasmic DNA polymerases.Cells of higher eukaryotes contain two DNAdependent DNA polymerases. One of these enzymes has a sedimentation coefficient of 6 -8 S and is present in the cytoplasm, the other sediments with 3.4 S and is largely, if not totally, confined to the nucleus. There is evidence that the cytoplasmic 6 -8-S enzyme increases in activity in the S-phase of proliferating cells, suggesting a role in replication; the nuclear 3.43 enzyme, in contrast, appears to be little regulated and its function is unknown [l -91.Earlier we have described the separation and partial purification of two DNA polymerases from yeast-cell extracts [lo]. Both enzymes, DNA polymerases A and B, sediment with about 8 S and differ from a third activity which is localized in mitochondria [l 11.As methods for the isolation of intact yeast nuclei in quantities sufficient for biochemical work have recently been developed [ 121, it has become possible to investigate which DNA polymerases are present in these isolated nuclei and in particular, whether a low-molecular-weight 3.4-S enzyme can be found. Using methods which sucessfully solubilize this polymerase from nuclei of mammalian cells [5] it was observed that no such enzyme can be isolated from yeast. DNA polymerases extractable from the nuclear fraction, in contrast, seem to be identical with those found in cell extracts and are of high molecular weight.Enzymes. DNA polymerase, deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase (EC 2.7.7.7); alcohol dehydrogenase, alcohol : NAD oxidoreductase (EC 1.1.1.1) ; malate dehydrogenase, L-malate : NAD oxidoreductase (EC 1.1.1.37). MATERIALS AND METHODS Isolation of Yeast NucleiA diploid wild-type strain of Sacchuromyces cerevisiae was used throughout this study. The cells grown in a medium containing 2% peptone (Difco), 1 % yeast extract (Difco) and 2% glucose to late log or stationary phase, harvested, washed and converted into spheroplasts as described using the snail-gut enzyme "Glusulase" from Endo Laboratories (USA.) [12]. For stationary-phase cells preincubation with EDTA and 2-mercaptoethanol was carried out for 1 h, for log-phase cells 30 min were found sufficient. Spheroplasts were washed with 1 M sorbitol and counted. Nuclei were prepared using a previously described procedure [ 121 which was slightly modified in order to obtain a preparation of nuclei containing less contaminating protein : The spheroplast pellet was suspended in 4 ml/g wet weight...

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“…This also makes it unlikely that the enzyme is mitochondria1 DNA polymerase. While the relationship, on interconversion, between At, A2 and C has been described [13] and the likely proteolytic origin of the 5.2 S enzyme, B, has been indicated both in mammalian cells [6,14] and in Drosophilu [27], the origin of enzyme D is uncertain. Properties such as closeness of their molecular weights, sensitivity to inhibition by N-ethylmaleimide, heat inactivation at 45 "C and lack of conversion to any other species by mild urea treatment, indicate that enzyme D more closely resembles enzyme C than A1 or Az.…”

Section: Discussionmentioning

confidence: 99%

“…When preparations of DNA polymerase-a from calf thymus, freed of DNA polymerase-p and terminal transferase, are fractionated on DEAE-cellulose, several peaks of polymerase-a activity can be detected using activated DNA in assays carried out at pH 7.8 [5,13]. From tissue frozen shortly after removal from the animal these are enzymes A1, A2 and C. A further enzyme B appears occasionally and is probably derived by proteolysis from enzyme C [6] (see also [14]). However, assay of the same DEAEcellulose profile with the synthetic template-initiator complex poly (dA) .…”

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confidence: 99%

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

Hesslewood

Holmes

Wakeling

et al. 1978

European Journal of Biochemistry

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The heterogeneity of calf thymus DNA polymerase-a has been further investigated. In particular, an enzyme (enzyme D) which exhibits higher activity on poly(dA) . (dT)lo (A : T = 20 : 1) compared with that on activated DNA, has been further purified and its properties compared with two other activities of the DNA polymerase-a fraction (enzymes A1 and C) which do not show a preference for poly (dA) . (dT)lo over activated DNA.As with A1 and C, enzyme D was shown to have many of the characteristic properties of DNA polymerase-a in that it is an acidic protein as judged by its binding to DEAE-cellulose, has a molecular weight of about 140000, does not use a poly(A) . (dT)Io template-initiator complex and is inhibited by N-ethylmaleimide. It exhibits anomalous gel filtration behaviour on Sepharose 6B and it binds relatively weakly to DNA-cellulose compared with DNA polymerase-p. The extreme sensitivity of enzyme D to inhibition by N-ethylmaleimide distinguishes it from A1 and C, as does its elution position from a DEAE-cellulose column. On the other hand enzymes C and D are readily inactivated by heating at 45 "C unlike enzyme Al.The possible interrelationships of the multiple activities of calf thymus DNA polymerase-a are discussed.Several different types of DNA polymerase activity have been described in mammalian cells [1,2]. Of these DNA polymerase-a is of particular interest since its activity is highest in situations where DNA synthesis is occurring [3]. A further point of interest concerns the heterogeneity of DNA polymerase-a reported in several instances, for example in calf thymus [4-71 rat liver and spleen [5], Chinese hamster cells [8], mouse myeloma [9-111 and baby hamster kidney (BHK) cells [12]. When preparations of DNA polymerase-a from calf thymus, freed of DNA polymerase-p and terminal transferase, are fractionated on DEAE-cellulose, several peaks of polymerase-a activity can be detected using activated DNA in assays carried out at pH 7.8 [5,13]. From tissue frozen shortly after removal from the animal these are enzymes A1, A2 and C. A further enzyme B appears occasionally and is probably derived by proteolysis from enzyme C [6] (see also [14]). However, assay of the same DEAEcellulose profile with the synthetic template-initiator complex poly(dA) . (dT)lo at pH 7.0 reveals a further

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Induction of DNA polymerase in mouse L cells

Cited by 219 publications

References 8 publications

X-ray Induced Activity of N-Ethylmaleimide-Sensitive DNA Polymerase in Chinese Hamster Cells

X-ray Induced Activity of N-Ethylmaleimide-Sensitive DNA Polymerase in Chinese Hamster Cells

Absence of a Low‐Molecular‐Weight DNA Polymerase from Nuclei of the Yeast, Saccharomyces cerevisiae

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

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