LINKAGE OF POLYNUCLEOTIDES THROUGH PHOSPHODIESTER BONDS BY AN ENZYME FROM ESCHERICHIA COLI* BY BALDOMERO M\I. OLIVERAt AND I. R. LEHMAN DEPARTMENT OF BIOCHEMISTRY, STANFORD UNIVERSITY SCHOOL OF MEDICINE, PALO ALTO, CALIFORNIA Communicated by Arthur Kornberg, March 20, 1967 There is now convincing evidence that recombinants are formed in genetic crosses by the breakage and joining of DNA molecules.' It is also likely that a similar breakage and rejoining occurs during "dark" repair of UV-irradiated DNA.2 Nucleases have been described which are, in principle, capable of catalyzing the specific breakage of DNA molecules.3 Recently, Gellert4 described an activity in extracts of E. coli which converted "hydrogen-bonded circles" of phage X DNA5 to a "covalently circular" form,6' I a reaction which may be analogous to the joining of DNA molecules. In this paper we describe the purification and some of the properties of an enzyme from E. coli that joins preformed polynucleotides in phosphodiester linkage. It does so only under the specific condition that the polynucleotides to be joined be part of a double-stranded structure. The purified enzyme requires for its action a diva- lent cation (1\Ig++ or Ca++) and a "cofactor," as yet unidentified. The cofactor is heat-stable; its activity is lost after treatment with Norit, and it cannot be re- placed by adenosine 5'-triphosphate (ATP) or any other known nucleoside tri- phosphate. The purified enzyme will also catalyze the formation of covalent circles from hydrogen-bonded circles of X DNA and would therefore appear to be similar, if not identical, to the activity described by Gellert. The substrate used in most of the studies to be described is a double-stranded homopolymer pair consisting of multiple dT8 units of 150-200 residues, hydrogen- bonded to a dA chain of approximately 3000 residues. The dT chains are labeled with p32 5'-phosphoryl termini so that the reaction is conveniently measured by the conversion of the labeled monoester phosphate to a form which is not susceptible to alkaline phosphatase. The reaction may be formulated as shown in Figure 1. Although the relationship of this enzyme, which we shall for convenience desig- nate as "polynucleotide-joining enzyme," to the act of genetic recombination is presently unknown, the availability, in reasonably purified form, of an enzyme that can link polynucleotides by a standard 3'5'-phosphodiester bond now makes it possible to explore recombinational and related phenomena at the enzymatic level. Experimental Procedure.-Materials: Unlabeled nucleotides and salmon sperm DNA were purchased from CalBiochem. HI-labeled phage X DNA (linear form) was isolated according to the method of Doherty and Hogness.9 It was converted to hydrogen-bonded circles as described by Wang and Davidson.10 3'-Deoxythymidine monophosphate (3'-dTMP) was synthesized according to Turner and Khorana.1 _yP32 ATP was prepared by the method of Glynn and Chappei1P2 as modified by Wu and Kaiser."3 Polynoucleotide kiniase was isolated as described by Richardson ;14 venom 5'-nucleotidase was purified by the method of Lehman, Roussos, and Pratt;'5 DNA polymerase from E. coli was plrepared according to Richardson et al.16 and micrococcal nulclease'7 was the gift of Dr. C. A. 1)ekker. The T4 phage-induced 3'-nucleotidase was isolated according to Becker and Ilurwitz.8 B. subtilis nuclease was prepared as described by Kerr, Chien, atid Lehman,19 and E. coli exo- nruclease I was purified by the method of Lehimami aiid Nimssbaum.20 E. coli alkaline phosl)hattse" was purchased fromi Worthington Biochemical Co. 1426 Downloaded by guest on September 29, 2021 VOL. 57, 1967 BIOCHEMISTRIYI: OLIVERA I ND LEHAJLAN 1427 5' P {P{P{'P{'P{PNV1~|Joinin{gAenz{{m' A A A A A A A A Cofcactor A A A A A A A A TT T T T T M ++ T T T T T T FIG. 1.-Synthesis of dT product b)y polylnucleotide-joining enzyme. Alethods: Polynucleotide concentrations are expressed as equivalents of nucleotide phosphoruis unless otherwise indicated; they were determined by measuring the absorbance at 260 mA and applying the extinction coefficients given by Riley, Mlaling, and Chamberlin.22 (a) Preparation of homopolymer substrates: The homopolymers dA and dT (mol wt, approxi- mately 106) were prepared as described by Riley et al.;22 the dT was degraded to smaller units by treatment with micrococcal nuclease. The progress of digestion by the nuclease was measured by treating small aliquots with an excess of polynucleotide kinase and yp32 ATP to determine the number of 5'-hydroxyl dT termini formed. The reaction was terminated when the average chain length was approximately 250 nucleotide residues. The 3'-phosphoryl termini of the dT were removed by treatment with bacterial alkaline phosphatase (10 units, 30 min at 370 for 0.3 /Lmole of dT). The phosphatase was inactivated by adjusting the pH to 10.5 with NaOH and heating in a boiling water bath for 30 min. The reaction mixture was brought to a pH of approxi- mately 8.5 by the addition of 1 M Tris-HCl, pH 7.5, then dialyzed against 0.01 M Tris-HCl, pH 7.5. The 5'-termini were labeled with p32 in a reaction with polynucleotide kinase as described by Richardson.14 The reaction mixture (5 ml) containing approximately 1 X 10-4 M dT (5 X 10-7 M in termini) was incubated at 370; 10-unit portions of polynucleotide kinase were added at 15-min intervals and the reaction was followed by determining acid-precipitable p32 until a limit was reached at between 60 and 90 min.23 The reaction mixture was dialyzed repeatedly against 1-liter changes of 1 M NaCl, containing 0.1 M Tris-HCl, pH 8.0, and 0.001 M ethylene- diaminetetraacetate (EDTA) until the dialysate contained fewer than 300 cpm per ml; this was followed by dialysis against two changes of 0.01 M Tris-HCl buffer, pH 8.0. The a' p32 dT was then centrifuged in an alkaline CsCl gradient, as described by Riley et al.,22 to separate it from the enzymes used in its preparation. After centrifugation, the dT solution was adjusted to pH 8.0-8.5 with 1 M Tris-HCl buffer, pH 7.5, and dialyzed against three 1-liter changes of 1 Al NaCl, containing 0.1 M Tris-HCl, pH 8.0, and 0.001 M EDTA, and three changes of 0.001 M Tris, pH 8.0. The 51_p32 dT prepared in this way had an average chain length of approximately 150 residues and a specific activity of 3-5 X 103 cpm per MSmole of 5'.p32 dT termini. (b) Assay of polynucleotide-joining enzyme: The standard assay for the polynucleotide-joining enzyme measures the conversion of the p32 5'-phosphoryl group of dT to a form insusceptible to the action of bacterial alkaline phosphatase. The incubation mixture (0.10 ml) contained 0.01 M Tris-HCl, pH 8.0, 0.002 M MgCl2, 0.001 M EDTA, 1.6 X 10 6 M dA and 1.6 X 10 6 M 5'- p32 dT (10-8 M in termini), 0.003-0.015 unit of enzyme and 2 ,A of boiled extract (20 mg protein/ ml; A260 = 389, prepared by heating a crude extract of E. coli (see below) for 5 min at 1000 then quickly chilling). After incubation of 30 min at 300, the reaction was terminated by boiling for 2 min. Five units of bacterial alkaline phosphatase24 were added and after thorough mixing, incubated for 15 min at 85°.25 The reaction mixture was chilled in ice and 0.2 ml of 1 N HCl, containing 0.1 AI Pi; 0.2 ml of 1 N HCl, containing 0.1 M PPi, 0.1 ml of bovine plasma albumin (1 mg/ml), and 0.2 ml of an acid-washed Norit suspension (20% packed volume) were added. The mixture was shaken, then filtered on a GF/C, 2.4-cm Whatman glass filter. The Norit was washed three times with 8 ml of 1 N HCl containing 0.1 A! Pi, and five times with 8 ml of 1 N IICI. The filter was dried and counted using a Nuclear-Chicago model 186 gas-flow counter equipped with a Micromil window. In the absence of enzyme, less than 0.5%7, of the input P32 remained Norit-adsorbable after phosphatase treatment. One unit of enzyme is defined as the amount catalyzing the transforma- tion of 1 MAmole of p32 5'-phosphoryl terminus of dT to a form insusceptible to bacterial alkaline phosphatase in 1 mill. The reaction rate was pioportional to the amount of enzyme added iii the range between 3 X 10-3 amd 15 X 1()-3 ,,,it. Downloaded by guest on September 29, 2021 1428 BIOCHEMISTRY: OLIVERA AND LEHMANV PROC. N. A. S. (c) Other methods: Protein concentrations were determined by the method of Lowry et al.26 In a few instances where concentrations were below 10 jg/ml, estimates were made by reading the absorbance at 280 mwu.2 Nucleotides were separated by paper electrophoresis at 40 in 0.02 Al sodium citrate buffer, pH 3.5, at a potential of 5000 v for 30 min. After electrophoresis, the paper was dried and the nucleotides were visualized under ultraviolet light. The paper was then cut into strips and the radioactivity determined in the gas-flow counter. When necessary, the nucleotides were eluted from the strips by soaking them in distilled water overnight at room temperature. H3-labeled samples were counted with a toluene-base scintillator using a Nuclear- Chicago model 724 liquid scintillation counter.