DNA ligase activity in UV-irradiated monkey kidney cells

DNA ligase was partially purified from normal and regenerating rat liver. Its structure was studied using the activity gel procedure that identifies the functional polypeptides. Two slightly different purification procedures were followed, leading to the isolation of one or two peaks (fractions A and B) of DNA ligase by hydroxyapatite chromatography. When analyzed on activity gels, all these enzyme fractions corresponded to a single active 130-kDa polypeptide both in normal and regenerating liver. A limited trypsin digestion of ligase fractions A and B gave rise to an identical pattern of smaller polypeptides of 110 kDa, 100 kDa and 75 kDa. Also storage at 4°C of fractions A and B produced smaller polypeptides of 110 kDa, 100 kDa, 85 kDa and 60 kDa, which were identical for the two fractions. Our results indicate that the same ligase polypeptide of 130 kDa can be isolated from stationary or regenerating rat liver cells. However, physiological or artifactual proteolysis during various purification procedures can lead to the isolation of two enzyme fractions with different chromatographic behaviour but with the same molecular mass.DNA ligase promotes the formation of a phosphodiester bond between 3'-hydroxyl and 5'-phosphoryl termini in a double-stranded DNA molecule and its activity is essential in joining DNA chains during replication, repair and recombination processes [I]. To fulfill these different functions a unique enzyme form has been found in T4 bacteriophage [2], E. coli [3] and yeast [4, 51. In mammalian cells two enzyme forms have been reported, DNA ligases I and 11, which seem to be involved in DNA replication and in DNA repair respectively [6]. Since these two activities can be distinguished from each other by their physical properties of molecular mass and sedimentation coefficient, ligases I and I1 activities can be separated in certain conditions either after chromatography or after sedimentation in velocity gradients of cellular extracts [7 -101. In fact after gel filtration analysis or velocity gradient sedimentations DNA ligase I has been described as a large enzyme with an apparent molecular mass ranging from 175 kDa to 220 kDa and a sedimentation coefficient of about 8 S, whereas ligase I1 has been described as a smaller enzyme of about 85 kDa and 4.5 S [6,7,10]. More recently it has been shown that, after NaDodS04/polyacrylamide gel electrophoresis, purified DNA ligases I and I1 from calf thymus present a single polypeptide with a molecular mass of 130 kDa and 67 kDa respectively [II, 121. DNA ligase I is supposed to be

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“…1 A, lane 2). For comparison, the activity gel of T4 DNA ligase is shown in lane 4, exhibiting the usual doublet of 60 kDa and 70 kDa [24,25].…”

Section: Correlation Of Dna Ligase Activity With a Polypeptide Of Higmentioning

confidence: 99%

Mammalian DNA ligase. Structure and function in rat-liver tissues

et al. 1987

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“…Some of the proteins induced in these * Corresponding author. cells, including DNA ligase (22) and 06-methylguanine-DNA-methyltransferase (19), are involved in DNA repair. Whether these proteins participate in a true mammalian SOS response remains to be determined, as does the biological role of these inducible responses.…”

mentioning

confidence: 99%

A constitutive damage-specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells.

Hirschfeld¹,

Levine²,

Ozato³

et al. 1990

Mol. Cell. Biol.

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Using a DNA band shift assay, we have identified a DNA-binding protein complex in primate cells which is present constitutively and has a high affinity for UV-irradiated, double-stranded DNA. Cells pretreated with UV light, mitomycin C, or aphidicolin have higher levels of this damage-specific DNA-binding protein complex, suggesting that the signal for induction can either be damage to the DNA or interference with cellular DNA replication. Physicochemical modifications of the DNA and competition analysis with defined substrates suggest that the most probable target site for the damage-specific DNA-binding protein complex is a 64'-(pyrimidine-2'-one)-pyrimiidine dimer: specific binding could not be detected with probes which contain -TT-cyclobutane dimers, and damage-specific DNA binding did not decrease after photoreactivation of UV-irradiated DNA. This damage-specific DNA-binding protein complex is the first such inducible protein complex identified in primate cells. Cells from patients with the sun-sensitive cancer-prone disease, xeroderma pigmentosum (group E), are lacking both the constitutive and the induced damage-specific DNA-binding activities. These findings suggest a possible role for this DNA-binding protein complex in lesion recognition and DNA repair of UV-light-induced photoproducts.

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“…Other proteins identified as having increased activity or expression as a response to DNA damage are DNA ligase [Mezzina and Nocentini, 1978; Sarasin, 19851, metallothionein [Lieberman et a1 1983;MacArthur et al, 1985; Angel et al, 19861, and H2 antigen (also induced by interferon) [Rahmsdorf et al, 19831. In addition, the enhanced synthesis of eight proteins was detected by two-dimensional gel electrophoresis following treatment of human diploid fibroblasts with UV, mitomycin C, or TPA [Schorpp et al, 19841.…”

Section: Other Inducible Functionsmentioning

confidence: 99%

“…However PA is also induced by tumor promoters such as TPA [Wigler and Weinstein, 19761, apparently more efficiently than by DNA-damaging agents. Although PA superficially resembles RecA protein in its properties of UV inducibility 106 Elespuru and protease activity, there is no evidence that it functions in an analogous manner in regulating gene expression.Other proteins identified as having increased activity or expression as a response to DNA damage are DNA ligase [Mezzina and Nocentini, 1978; Sarasin, 19851, metallothionein [Lieberman et a1 1983;MacArthur et al, 1985; Angel et al, 19861, and H2 antigen (also induced by interferon) [Rahmsdorf et al, 19831. In addition, the enhanced synthesis of eight proteins was detected by two-dimensional gel electrophoresis following treatment of human diploid fibroblasts with UV, mitomycin C, or TPA [Schorpp et al, 19841.…”

mentioning

confidence: 99%

Inducible responses to dna damage in bacteria and mammalian cells

Elespuru¹

1987

Environ and Mol Mutagen

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INTRODUCTIONThis review summarizes current knowledge of the DNA damage-inducible SOS response in E. coli, with an emphasis on the historical development of experimentation in this area. Evidence for the existence of analogous responses to DNA damage in other bacteria, lower eukaryotes, and mammalian cells is also presented. The mechanistic basis in E. coli for one of the SOS responses, mutagenesis, is currently a subject of intensive study in many laboratories, and recent developments in this area are explored in greater detail. Mutagenesis in mammalian cells, which does not appear to conform to the SOS model, is the subject of a forthcoming review [Rossman and Klein, in press].For more detailed treatments of various aspects of the SOS response in bacteria and mammalian cells, other recent reviews are recommended: the SOS response in E. coli [Witkin, 1976;Oishi et al, 1981;Little and Mount, 1982; Gottesman, 1984; Walker, 19841; pKMlOl [Strike and Lodwick, 19871; lambda prophage induction [Roberts and Devoret, 1983; Elespuru, 19841; DNA repair [Hanawalt et al, 1979;Defais et al, 1983; Walker et al, 19851; the SOS response in mammalian cells [Radman, 1980;Sarasin, 1985;Rossman and Klein, 1985; Lambert and Garrels, 19861. The contents of this publication do not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.By acceptance of this article, the publisher or recipient acknowledges the right of the US Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article.Published 1987 by Alan R. Liss, Inc. Elespuru THE SOS RESPONSE IN E. coliObservations of apparently unrelated phenomena (virus reactivation, mutagenesis, prophage induction, filamentation) in different genetic backgrounds in E. coli led to a unifying hypothesis called the "SOS response" [Radman, 19751. The "SOS" hypothesis suggested "the existence of a DNA repair system which is normally repressed but which is induced by DNA damage". Two early concepts were critical to the development of the SOS model. One was that mutagenesis and repair of DNA damage depended on an inducible cellular process [Sedgewick, 1975; Witkin, 19761. This concept developed from the experiments of Weigle and others, which showed that survival and mutagenesis of UV-irradiated lambda phage depended on the irradiation of the host E. coli and was inhibited by the presence of chloramphenicol Weigle, 1953; Defais et al, 19761. The second concept, of coordinate regulation of responses to DNA damage, arose from genetic experiments. Mutations in certain genes (rec and lex) resulted in the abolition of all of the known responses to genetic damage under study (Weigle reactivation, mutagenesis, lambda prophage induction, filamentation) or to their temperature-dependent expression witkin, 1967, 1969; Castellazzi et al, 1972; Ishii and Kondo, 19751. The abolition of "Weigle reactivation" (enhanced survival o...

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DNA ligase activity in UV-irradiated monkey kidney cells

Cited by 39 publications

References 22 publications

Mammalian DNA ligase. Structure and function in rat-liver tissues

Mammalian DNA ligase. Structure and function in rat-liver tissues

A constitutive damage-specific DNA-binding protein is synthesized at higher levels in UV-irradiated primate cells.

Inducible responses to dna damage in bacteria and mammalian cells

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