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DNA LIGASE---MAMMALIAN
333
[27] DNA Ligase from Rabbit Tissues 1
By TOMAS LINDAHL Assay Method Principle. The assay method is that of Weiss et al. 2 with minor modifications. It measures the conversion of ~2P-labeled 5' ends in nicked DNA molecules to a form resistant to alkaline phosphatase. Reagents. The reaction mixture (total volume 0.3 ml) contains:
Tris.HC1, 70 mM, pH 7.5 MgC12, 10 mM 2-Mercaptoethanol, 10 mM K2HPO~, 5 mM ATP, 0.5 mM 5'-[32P]Phosphoryl DNA, 1 ~g (see below, preparation of substrate) Enzyme, 0.1-0.5 unit The other assay reagents are: Carrier DNA (heat-denatured calf thymus DNA), 0.5 mg/ml Trichloroaeetic acid, 0.8 M HC1, 10 mM N a 0 H , 0.1 M Tris.HC1, 0.2M:l.1 M Escherichia coli alkaline phosphatase, 2 mg,/ml in 0.2 M NaC1, 10 mM Tris, pH 8.0 Procedure. The mixture is incubated at 37 ° for 15 minutes, and then chilled to 0 °. To each sample, 0.2 ml carrier DNA solution and 0.5 ml cold 0.8 M trichloroacetic acid are added. After centrifugation at 10,000 g for 10 minutes, the supernatant is removed, and the precipitate is resuspended in 2 ml of 10 mM HC1 with a thin glass rod. After recentrifugation the supernatant is again discarded, and the precipitate is dissolved in 0.5 ml of 0.1 M NaOH. The solution is brought to pH 8.0 ___0.5 with 50/A of 0.2M Tris-l.1 M HC1. Alkaline phosphatase (10 t~g) is added, and the mixture is incubated for 30 minutes at 37 ° and then for 30 minutes at 65 °. After chilling to 0 °, 0.5 ml of cold 0.8 M trichloroacetic acid is added, and the precipitate is collected on a membrane filter, washed with 1T. Lindahl and G. M. Edelman, Proc. Nat. Acad. Sci. U.S. 61, 680 (1968). 2B. Weiss, A. Jacquemin-Sablon, T. R. Live, G. C. Fareed, and C. C. Richardson, J. Biol. Chem. 243, 4543 (1968).
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several portions of 0.4 M trichloroacetic acid, and dried; its radioactivity is subsequently determined. Unit. One unit of enzyme converts 1 pmole of ~2p to an alkaline phosphatase-resistant form in 15 minutes. Preparation o] Substrate. Native calf thymus DNA, 2 mg/ml in 50 mM Tris.HC1, pH 7.5, 3 mM MgC12, is partially degraded by treatment with 0.02 .~g/ml pancreatic DNase for 30 minutes at 25 °. An aliquot of this DNA solution should contain 2-4% acid-soluble material. A 0.05 volume of 0.2 M EDTA, pH 8, is added, and the mixture is heated to 65 ° for 5 minutes to inactivate the DNase, followed by dialysis overnight against 20 mM NaC1, 10 mM Tris.HC1, pH 8.0. The DNA solution is brought to 65 ° and incubated with 30 ~gc/ml alkaline phosphatase for 20 minutes. After cooling, the reaction is stopped by the addition of 0.1 volume of 20 mM K~HP04. The phosphatase-treated DNA is subsequently labeled with a~p by incubation in a reaction mixture containing the DNA (1 mg/ml), 70 mM Tris.HC1, pH 7.5, 10 mM MgC12, 5 mM K2HP04, 10 mM 2-mercaptoethanol, 5 X 10-SM [ ~ - - ~ P ] A T P (2 X 109 cpm/t~mole), and polynucleotide kinase ~ (50 units/ml). The reaction is stopped after 20 minutes by adding 0.1 volume of 0.5 M K~HPO~, 0.2 M EDTA, pH 8, followed by heating to 50 ° for 5 minutes to inactivate the polynucleotide kinase. The DNA substrate is dialyzed for 4 days against several changes of 1 M NaC1, 50 mM K~HPO~, 1 mM EDTA, pH 8.0, to remove excess ATP, and stored at 0 °. The final preparation still contains alkaline phosphatase; this enzyme is not active in the presence of Pi, but may be removed by extraction in an aqueous two-phase system.1 A typical substrate preparation contains 5 ,~Ci of 82p per milligram of DNA, with 20% of the label at reparable single-strand breaks. It should be noted that contaminating activities in the enzymes used as reagents may interfere during the preparation of the substrate. Such activities include: (a) DNase in E. coli alkaline phosphatase. Commercially obtained preparations of alkaline phosphatase often contain substantial amounts of DNase activity. The DNase is inactivated by dialysis of a concentrated solution of alkaline phosphatase against 0.2M Tris.HC1, 10 m M M MgC12, pH 8.0, followed by heating at 95 ° for 5 minutes; (b) DNase in polynucleotide kinase. Traces Of DNase activity in preparations of this enzyme may be removed by gel filtration on Sephadex G-200, polynucleotide kinase is eluted before the DNase activity. This additional purification procedure is most conveniently introduced as the next to last step during the purification of the enzyme; (c) DNA ligase in polynucleotide kinase. A contamination with the phage-induced DNA ligase may s C. C. Richardson,Proc. Nat. Acad. Sci. U.S. 54, 158 (1965).
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DNA LIGASE--MAMMALIAN
335
give a substrate containing significant amounts of phosphatase-resistant 32p, however, this can be avoided by using E. coli infected with a ligasenegative T4 amber mutant as the source of polynucleotide kinase. Purification Procedure All operations are performed at 0°-4 °. Centrifugations are for 15 minutes at 12,000 g unless otherwise stated. All solvents contain 10 mM 2mercaptoethanol. Crude Extract. Frozen spleens from mature rabbits may be obtained commercially (Pel-Freez Biologicals, Inc., Rogers, Arkansas). Fifty grams of frozen spleens are partially thawed and added to 250 ml of 0.1 M NaC1, 50 mM Tris-HC1, 1 mM EDTA, pH 7.4. The tissue is cut in small pieces, and the cells are disrupted in portions by a few strokes in a glass hand homogenizer. The homogenate is stirred gently for 1 hour and centrifuged at 25,000 g for 30 minutes to remove debris and fat (fraction I). Ammonium SulJate Fractionation. Fraction I is diluted with an equal volume of the extracting solution. Solid (NH4)2S04 is then added to 40% saturation. After 30 minutes the precipitate is removed by centrifugation. To the supernatant solution additional (NH4)2S04 is added to a final concentration of 65% saturation; 30 minutes later the precipitate is recovered by centrifugation, redissolved in 50 ml of 20 mM potassium phosphate, 1 mM EDTA, pH 7.2, and dialyzed against 10 liter of the same buffer for 8-10 hours. A light precipitate, formed during the dialysis, is removed by centrifugation (fraction II). Phosphocellulose Chromatography. Fraction II is applied to a column of phosphocellulose (2 X 20 cm), equilibrated with 20 mM potassium phosphate, 1 mM EDTA, pH 7.2. The column is washed with 25 ml of the same solvent, and then with 250 ml of 50 mM KC1, 20 mM potassium phosphate, 1 mM EDTA, pH 7.5. The enzyme is subsequently eluted with 0.5 M KC1, 20 mM potassium phosphate, 1 mM EDTA, pH 7.5; fractions of A28o > 0.5 are recovered and pooled (fraction III). Hydroxy.apatite Chromatography. Fraction III is dialyzed against 4 liters of 50 mM potassium phosphate, pH 7.5, for 4 hours and applied to a column of hydroxyapatite (2)< 6 cm) equilibrated with the same buffer. The column is then washed with 20 ml of this buffer, followed by 50 ml of 0.15 M potassium phosphate, pH 7.5. The enzyme is eluted with 0.5M potassium phosphate, pH 7.5; fractions of A2so > 0.5 are recovered, pooled, and dialyzed against 4 liters of 0.1M potassium phosphate, pH 7.5, for 10 hours (fraction IV). Alumina C~ Gel Fractionation. To determine the highest buffer concentration at which the enzyme is adsorbed to alumina C~ gel, a pilot
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experiment is performed in the following way: Twelve 0.05-ml aliquots of fraction IV are adjusted to various concentrations of potassium phosphate between 0.05 and 0.16 M by addition of water or 0.5 M phosphate buffer. Then 0.01 ml of a 2% suspension of alumina C-/ gel in 0.1 M potassium phosphate, pH 7.5, is added to each sample. After mixing, the samples are centrifuged, and the A2so and enzymatic activity of each supernatant is measured. More than 80% of the ligase activity is usually bound to the gel when 30% of the total protein is adsorbed. Fraction IV is adjusted to the highest phosphate concentration at which >80% of the enzyme will adsorb to alumina C~ gel. Then 0.2 volume (of the volume of fraction IV before the adjustment) of a 2% alumina C~ gel suspension in 0.1 M potassium phosphate, pH 7.5, is added; the mixture is stirred gently for 15 minutes. After centrifugation the supernatant is discarded. The C~ gel is washed once with 10 ml of a phosphate buffer of the same concentration as that of the discarded supernatant. The enzyme is subsequently eluted from the gel with 2 ml (first eluate) and 1 ml (second eluate) of 0.5M potassium phosphate, pH 7.5. The two eluates are pooled and dialyzed overnight against 0.4 M KC1, 50 mM Tris.HC1, 1 mM EDTA, pH 7.2 (fraction V). Cold glycerol is added to a final concentration of 30%, and the enzyme solution is stored at --10% A summary of the purification procedure is given in the table. Fraction V is labile and loses 50% of its activity in 1-2 weeks at --10 °. All fractions of the enzyme have a half-life of about 3 days at 0 °, and the purification should therefore be done as rapidly as possible. Fraction V may contain traces of DNA polymerase and exonuclease [for poly (dA-dT)] activities. No preparations have had detectable endonuclease activity.4 PURIFICATION OF DNA LIGASE FROM RABBIT SPLEEN
Fraction I. II. lII. IV. V.
Crude extract Ammonium sulfate Phosphocellulose Hydroxyapatite Alumina C~ gel
Volume (ml)
Total protein (mg)
Specific activity (ligase units/mg)
Total activity (ligase units)
262 76 68 10 3
9400 2100 150 38 12
0.9 3.4 36 65 130
8700 7200 5400 2500 1600
' Endonuclease activities for native and denatured DNA have been assayed according to E. P. Geiduschek and A. Dtmiels, Anal. Biochem. 11, 133 (1965).
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DNA L I G A S E - - M A M M A L I A N
337
Properties of the Purified Enzyme Specificity. The enzyme repairs DNA having single-stranded interruptions with juxtaposed 3'-hydroxy- and 5'-phosphate termini. The average chain length in such a DNA preparation is thus increased. At the site of joining, a 3'-5' phosphodiester bond is formed. Requirements. The ligase has a pH optimum at 7.5-8.0 in Tris.HC1 buffer. The presence of a divalent metal ion is necessary for activity: Mg ~÷ is more effective than Mn 2+, and Ca 2÷ is completely ineffective. The optimal Mg 2÷ concentration is 10 mM. In the absence of 2-mercaptoethanol the ligase is very labile, and it is then completely inactivated by low concentrations of p-hydroxymercuribenzoate. The enzyme has an absolute requirement for ATP; the Km for ATP in the ligase reaction is 2 X 10-~ M. DPN, GTP, and TTP cannot replace ATP as a cofactor. With high concentrations of chromatographically purified dATP, some cofactor activity is observed; however, it is not known whether this activity is due to remaining traces of ATP. When ATP and dATP are both present in the reaction mixture, the enzyme is not detectably inhibited by a 10-fold excess of the latter compound. Size. The Stokes radius of the ligase activity has been estimated by analytical gel filtration to be 39 -~. For a typical globular protein, this corresponds to a molecular weight of 95,000 ___ 15,000. Mechanism of Reaction. The partially purified DNA ligase preparation obtained by this procedure catalyzes ATP-PP~ exchange in the absence of DNA. The exchange activity and the ligase activity show similar requirements and similar fractionation properties in the final purification steps. This suggests that the mammalian DNA ligase works by a similar mechanism as the microbial enzymes of this type, with the formation of an enzyme, adenylate intermediate. Subcellular Localization. In mammalian cells, ligase activity is primarily found associated with cell nuclei. The enzyme shows a similar subcellular distribution to that of DNA polymerase and DNase IV in that it is not firmly bound to the nuclei, instead it leaks out into the cytoplasm to some extent during the isolation of the subcellular fractions. The ATP-dependent DNA ligase appears to be the major, if not the only, enzyme of this type occurring in the cell nuclei or cytoplasm of normal rabbit cells. No indication has been obtained for the simultaneous existence in such material of a DPN-dependent DNA ligase similar to that in E. coll. Cellular Distribution. In the rabbit, the highest levels of DNA ligase activity are found in extracts from myeloid and lymphoid tissues (bone marrow, spleen, thymus, embryonic liver), but enzyme activity also is present in extracts from other rapidly growing cells (intestinal epithe-
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lium) and in very small amounts in liver extracts. The ligase activities from the different tissues show similar fractionation properties and are ATP-dependent; it seems likely that the same enzyme occurs in several tissues. A similar amount of ligase activity to that in rabbit lymphoid tissue is observed in extracts from mouse myeloma tumors. Extracts from spleens of larger animals (pig, calf, sheep) also contain detectable DNA ligase activity, but apparently the enzyme is present in relatively lower amounts than in rabbit spleen extracts.