MAPPING THE 5'-TERMINAL NUCLEOTIDES OF THE DNA OF BACTERIOPHAGE X AND RELATED PHAGES* BY RAY WUt AND A. D. KAISER DEPARTMENT OF BIOCHEMISTRY, STANFORD UNIVERSITY SCHOOL OF MEDICINE, PALO ALTO, CALIFORNIA Communicated by Arthur Kornberg, October 28, 1966 The two ends of a molecule of X DNA have a structure which enables them to join together to form circular monomers1-' or linear dimers, trimers, etc.' Many, if not all, temperate bacteriophages have cohesive ends including coliphages 480,4 21, 186, 424, 434,5 and P2.6 The thermal reversibility of cohesion1'7 and the response of the ends of X DNA to Escherichia coli DNA polymerase and exonuclease III8 suggest that each end has a short single strand, about 15 nucleotides long, which protrudes beyond a long double-stranded interior of about 50,000 nucleotide pairs. The single strand at one end is judged to be complementary to the single strand at the other end because cohesion occurs only between a "left" end and a "right" end.31 Figure 1 shows how a molecule with complementary single strands protruding from its ends can form a circular molecule by base-pairing between the two single strands. left half right holf' sus sus 31 HO I W) V oCO = heov4 Strand I i qht strand FIG. L.-Structure of X DNA and formation of circular molecules. The structure of the 5'- termini is represented diagrammatically;88MMSA, SU8SB, and ix are genetic markers;7 other sym- bols are described in the text. Soon after infection of a sensitive or a lysogenic bacterium, the two ends of the DNA molecule of an infecting phage cohere to each other and the single-strand inter- ruptions are closed by covalent bonds.9' 10 Thus cohesive ends provide a mechanism by which DNA molecules can be joined transiently or permanently. The occur- rence of cohesive ends in many different temperate phages and the possibility that cohesion may provide a general mechanism for the joining of two DNA molecules prompted us to initiate a study of the sequence of bases in cohesive ends. We wish to report here the identification of the 5'-terminal nucleotides of the DNA of phages X, 480, 21, and 186. The method for identifying 5'-termini is that of Richardson.11 It consists in attaching a P"2-phosphoryl residue to the 5'-OH terminus of a native DNA molecule in a reaction catalyzed by polynucleotide ki- nase,12, 13 then hydrolyzing the DNA to yield 5'-mononucleotides, and finally de- termining which nucleotides carry p32. Since DNA isolated from bacteriophage X is biologically active."4 it has also been possible to orient the two 5'-termini with respect to the genetic linkage map. Materials.-The strains of phage employed were X ind- C1 857,15 21gp,1' ,80,17 and 186p.6 Stocks of X and 186 were prepared by thermal induction. Eacherichia coli K12, strain W3350 (X ind- 170 VOL. 57, 1967 BIOCHEMISTRY: WU AND KAISER 171 C, 857) or W3350 (186p), was grown at 350 with aeration in tryptone broth to an optical density of 1.0 (at 590 mjs), induced by heating to 450 for 10 min, then incubated at 370 with aeration until maximal lysis occurred as indicated by a minimum optical density. Stocks of 21 were pre- pared by ultraviolet light induction of C600 (21gp). Stocks of 480 were prepared by infection of W3350 grown in tryptone broth. Phage were purified and the DNA extracted according to the procedure of Kaiser and Hogness,'4 except that phenol was saturated with Tris buffer (0.01 M) and adjusted to pH 7.1 before use. At least 80% of the single strands were free of breaks in the X DNA preparations, as judged by zone sedimentation in 0.1 M NaOH and 0.9 M NaCl.18 The preparations of 21, ,80, and 186 DNA were sedimented in a sucrose gradient at neutral pH; more than 75% of the UV-absorbing material sedimented as a single peak of double-stranded DNA. _y-P32-adenosine 5'-triphosphate(pyro) (ATP) was prepared according to the procedure of Glynn and Chappell.19 Polynucleotide kinase was purified according to the procedure of Richardson,'3 modified by adding ATP (to 0.1 mM) to the solutions used after the ammonium sulfate step to stabilize the enzyme and by an additional O-(diethylaminoethyl) (DEAE)-cellulose column step to remove excess nucleic acid. The latter was carried out by diluting the ammonium sulfate fraction with 4 vol of water, and applying the solution to a 6 cm2 X 12 cm DEAE-cellulose column. Most of the kinase activity (60-80%) eluted with 50 ml of 0.01 M potassium phosphate, pH 7.5, 0.01 M 2-mercaptoethanol. The enzyme was concentrated by precipitation with 2.9 gm of ammonium sulfate per 10 ml of eluate, dissolved in 10 ml of the elution buffer, diluted with 70 ml of 0.005 M 2-mercaptoethanol, and processed on DEAE and phosphocellulose columns as described by Richardson." The phosphocellulose fractions were concentrated by precipitation with 3.7 gm of ammonium sulfate per 10 ml of eluate, and the precipitate was dissolved in 2 ml (per 10 ml of eluate) of 0.02 M Tris buffer (pH 7.5) containing 0.01 M 2-mercaptoethanol. The product had a specific activity of 30-50,000 units/mg and contained no nuclease activities detectable by the following tests. Thirty units of kinase were incubated for 60 min with X DNA under the condi- tions described below for the phosphorylation of DNA. The pattern obtained when the product was sedimented as a band in alkaline solution was the same as that given by the DNA before exposure to the kinase. P32-labeled E. coli DNA (20 miumoles, 8 X 106 cpm/,smole) was incu- bated with 5 units of kinase for 30 min, no increase (less than 0.05%) in acid-soluble P32 was found. Venom phosphodiesterase (Worthington Biochemical Corp.) was further purified to remove small amounts of 5'-nucleotidase activity.20 Pancreatic DNase and E. coli alkaline phosphatase (chromatographically purified) were products of the Worthington Biochemical Company. Methods.-Phosphatase treatment of DNA: The incubation mixture contained 0.1 M Tris buffer (pH 8.0), 0.2-0.8 mM DNA (expressed as nucleotide equivalents), 1mM ethylenediamine- tetraacetate (EDTA) (to inhibit contaminating nucleases in the phosphatase preparation), and 10-30 ug of alkaline phosphatase/ml. After incubation for 60 min at 370, the reaction mixture was extracted twice with buffer-saturated phenol and dialyzed for 3 days (with two changes) against 100 vol of Tris buffer (0.01 M, pH 8.0) containing 1 mM EDTA. This treatment intro- duced no single-strand breaks detectable by band sedimentation in alkali. Phosphorylation of DNA: The reaction mixture contained 0.07 M Tris buffer, pH 7.6, 12 mM MgCl2, 10 mM 2-mercaptoethanol, 0.12-0.3 mM DNA (or phosphatase-treated DNA), 0.03 mM y-P32-ATP (specific activity 1-4 X 109 cpm/lAmole), and 30 units of polynucleotide kinase/ml added in two equal portions at 0 and 30 min. After incubation at 370 for 60 min, EDTA was added to 20 mM and the mixture was chilled and passed through a 1 X 35-cm column of Sephadex G50 (100-300-,u particle size) to remove the unreacted -y-P31-ATP, and then sedimented in a gradient of 5-20% sucrose containing 1 M NaCl, 0.01 M Tris buffer, pH 7.1, and 0.001 M EDTA at 60,000 X g for 12 hr. In some experiments the mixture was layered directly on a sucrose gradient and centrifuged. Intact DNA molecules were collected in fractions 5 through 8 out of a total of 17 fractions. Of the input DNA, 80% was usually recovered as intact molecules and the ratio of P32 to A260 was constant in these fractions. The p32 content of the phosphorylated DNA isolated in the peak fractions was determined after the addition of salmon sperm DNA as carrier and precipitation of the DNA by 0.5 N perchloric acid. The precipitated DNA was dissolved in 2 ml of 0.05 N NaOH and reprecipitated with perchloric acid; the precipitate was then col- 172 BIOCHEMISTRY: WU AND KAISER PROC. N. A. S. lected on a glass filter, washed, dried, and the radioactivity determined. The specific radioactivity of the phosphorylated DNA was calculated from the optical density of the DNA of the sucrose fractions and its content of acid-precipitable p32, taking the molar absorbance at 260 m/A as 6,600. 14 Hydrolysis of purified DNA to nucleoside-5'-monophosphates: Phosphorylated DNA was pre- cipitated with 10% trichloroacetic acid and sedimented. The precipitated DNA was washed twice with 3-ml portions of cold 80% ethanol and resuspended in 0.1 ml of water. The DNA was then digested by the addition of 0.05 ml of 0.1 M Tris acetate (pH 8.0), 0.01 ml of 0.1 M magnesium acetate, and 0.01 ml of pancreatic deoxyribonuclease (2 mg/ml), and by incubation at 370 for 1 hr. The reaction mixture was centrifuged and the clear supernatant solution (con- taining partially digested DNA) was transferred to another test tube. It was further digested after the addition of 0.03 ml of 0.1 M glycine acetate (pH 9.0) and 0.01 ml of venom phospho- diesterase (1500 units/ml), and by incubation at 37° for 45 min. Essentially all of the radio- activity was converted to mononucleotides as judged by chromatography.