Proc. Nat. Acad. Sci. USA Vol. 70, No. 8, pp. 2238-2242, August 1973 The Synthesis and Enzymatic Polymerization of Nucleotides Containing Mercury: Potential Tools for Nucleic Acid Sequencing and Structural Analysis (acetoxymercuration/mercaptans/Escherichia coli/polymerases) R. M. K. DALE, D. C. LIVINGSTON*, AND D. C. WARD Department of Molecular Biophysics and Biochemistry, Yale University, 333 Cedar Street, New Haven, Connecticut 06510 Communicated by Frederick M. Richards, May 1, 1973 ABSTRACT A simple acetoxymercuration reaction for also circumvent most of the present problems of preparing introducing covalently bound mercury atoms into nucleo- heavy-atom polynucleotide derivatives. The current methods tides is described. The 5-mercuriacetate derivatives of UTP, CTP, dUTP, and dCTP, as well as the 7-mercuriace- of attaching electron-dense atoms onto polynucleotides, in- tate derivative of 7-deazaATP, have been prepared by this volving chemical modification of preexisting DNA or RNA procedure and tested as substrates for nucleic acid poly- polymers (5, 6), often give unstable products, incomplete sub- merases. These nucleotides, in the absence of added mer- stitution, or fragmentation of the polynucleotide chain (7). captan, are not polymerized and in most instances are of a simple method of preparing polymers potent enzyme inhibitors. However, conversion of these The availability mercuriacetates to mercurithio compounds in situ by the with specific heavy-atom base labels should make sequence addition of one of various mercaptans, yields nucleoside analysis limited only by the resolving power of the electron triphosphates that are excellent substrates for all poly- microscope. merases tested: Escherichia coli and T7 RNA polymerases, Metallonucleoside derivatives can also be readily used DNA polymerase I of E. coli, DNA polymerase of avian myeloblastosis virus, and calf-thymus terminal deoxy- for analysis of protein or polynucleotide structures, as nucleotidyl transferase. By varying the mercaptan used to well as investigation of specific protein-polynucleotide promote syntheses it is possible to access certain structural complexes. In addition to the obvious utility of heavy-atom limitations in the enzyme's nucleoside triphosphate bind- analogues as isomorphous replacements in crystallographic ing site. These mercurinucleotides appear to have a di- designed compounds should provide versity of potential applications: (1) as heavy-atom re- studies, appropriately agents for crystallographic and microscopic studies; (2) electron spin resonance or affinity probes for investigation as affinity probes for enzymes sensitive to sulfhydryl of macromolecular structure in solution. modification; (3) as steric probes of substrate-binding With objectives like those outlined above in mind, we have sites on enzymes; and (4) as reagents for forming covalent undertaken the preparation of various metallonucleotides. protein-polynucleotide complexes. In this report, we describe the synthesis and some properties Numerous nucleoside analogues have been synthesized during of nucleotides containing covalently bound mercury atoms. the past decade, mainly for examination of their chemother- METHODS AND MATERIALS apeutic activity. One group of compounds that appears to 5'-triphosphates is the metallonucleosides. Although The standard ribo- and deoxyribonucleoside have escaped attention were purchased from P. L. Laboratories, while deoxyuridine likely to be of limited potential as chemotherapeutic agents, were obtained from Sigma. and coenzyme derivatives of and deoxyuridine 5'-triphosphate nucleotide, polynucleotide, Radioactive nucleotides were products of New England Nu- such compounds could provide a set of useful tools for various the below. clear Corp. Tubercidin (7-deazaadenosine), generous biochemical studies, a few of which are outlined The Upjohn Co., Kalamazoo, Mich., technology has progressed to gift of Dr. G. Fonken, Recent electron microscope was converted to the 5'-triphosphate as described (8). the state where single heavy-atoms have been visualized both ribo- reduc- The pyrimidine nucleoside triphosphates (in (1-4). Although further refinements are needed (e.g., and the 5'-triphosphate of 7-deaza- noise in grids), microscopic and deoxyribo-series) tion of the background support adenosine were converted to the corresponding mercuriacetate sequencing of polynucleotides is potentially feasible. Metal- The nucleo- to their compounds by the following general procedure. lonucleoside triphosphates that are able replace was dissolved in 5.0 ml of 0.5 M as in vitro side triphosphate (1 mmol) natural triphosphate counterparts quantitatively sodium acetate buffer (pH 5.0), and mercuric acetate (5.0 substrates for nucleic acid polymerases would yield polymeric mixture was then heated The mmol) was added. The reaction products suitable for such sequence analysis. enzymatic after was diluted 10- stable metallonucleotides would at 500 for 3 hr. The mixture, cooling, polymerization of chemically to 20-fold with water and applied to a column (110-ml) of DEAE-cellulose (bicarbonate form). The column was washed Abbreviations: dUMP-HgX, dUTP-HgX, and CTP-HgX, the 5- exhaustively with water (about 4 liters) to remove excess mer- mercuriacetate derivatives of dUMP, dUTP, and CTP. 7- curic salts before the products were eluted with a linear gradi- DeazaATP-HgX the 7-mercuriacetate derivative of 7-deazaATP of bicarbonate (0-0.5 M), UTP-HgSR, etc. the ent (2 liters) triethylammonium (tubercidin 5'-triphosphate). dUTP-HgSR, pH 7.5. Fractions containing the mercurinucleoside triphos- 5-mercurithio derivatives of dUTP and UTP prepared by reacting After the appropriate mercuriactetate compound with an excess of a phate were pooled and desalted by rotary evaporation. mercaptan. several washes with methanol the nucleotide was dissolved to and stored at -20°. Conversion * Permanent address: Dept. of Chemistry, Imperial Cancer Re- in water, adjusted pH 7.0, search Fund, LincoIns Inn Fields, London WC2, England. to the mercurinucleotide was in all cases quantitative, al- 2238 Downloaded by guest on October 1, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Polymerization of Nucleotides Containing Mercury 2239 though heating at 500 did cause a small degree of hydrolysis 0 NH2 NH2 HgX to the mono- and HqX corresponding diphosphates (which were HINA NX gHX readily removed chromatographically). Spectral, chromato- were used to graphic, and electrophoretic analyses demon- N 00'."N %NN strate that the mercuri-products are free from unmodified HOCH$HO HOC starting material. Radiolabeled mercurinucleoside triphosphates were pre- OH OH OH OH OH OH pared as above with [20'Hg]mercuric acetate (New England (H) (H) Nuclear Corp.). The standard conditions were: 5 pmol of I mE nucleoside triphosphate in 0.05 ml of acetate buffer and 25 FIG. 1. The structure of 5-mercuriuridine (deoxyuridine), ,Amol of [20'Hg]mercuric acetate (20 Ci/mol). (I); 5-mercuricytidine (deoxycytidine), (II); and 7-mercuri-7- Mid-log-phase E8cherichia coli B cells were obtained from deazaadenosine, (1I). The counterion, X, varies according to Grain Processing Corp. E. coli DNA polymerase I (Fraction the ionic environment but is normally acetate, carbonate, or VII) was prepared as described by Jovin et al. (9) with the chloride. In the presence of mercaptans the compounds are con- exception that the final Sephadex G-100 gel-filtration column verted to the corresponding mercurithio compounds (nucleoside- was eluted with phosphate buffer (pH 7.0) containing no 2- HgSR). The spectral properties of the carbonate salts at pH 7.5 mercaptoethanol. E. coli B RNA polymerase, purified by the are: I, X.., 267 nm (e = 10,100), X1min, 242 (e = 3600); II, Asxs, procedure of Burgess was the of R. and 275 (E 9200), Xmin 255 (e = 5600); m, Xm, 278 (e 12,000), (10), gift Ludwig Xmin, 244 (e = 2900). Although the absorption maximum is un- R. Condit. Commercial preparations of this enzyme (Grand affected by the nature of the counter ion present, the wavelength Island Biological Co.) gave similar results. The T7 RNA and extinction of the absorption minimum does vary somewhat. polymerase and T7 DNA were the generous gifts of Drs. E. Niles and W. Summers. Calf-thymus terminal deoxynucleo- strates (17, 19), provided that the substituent is not charged tidyl transferase (11) was kindly supplied by Dr. R. Ratliff, at the pH of the enzyme incubation. Los Alamos, Calif. In another communicationf we describe properties of The RNA-dependent DNA polymerase of avian myelo- 5-methylmercurithio- and 5-trimethyllead pyrimidine nucleo- blastosis virus was prepared from a viral preparation sup- side compounds. Since the synthetic routes to these com- plied by Dr. J. Beard, Duke University, as described by pounds were rather extensive, simpler approaches were Green et al. (12). For enzyme assays the virus solution was considered. Methods of preparing mercury-carbon organo- preincubated in 0.5% NP-40 detergent (Shell) at 40 for 30 mercurials by hydrogen substitution have been known since min before addition to the polymerization mixture. the mid-1800s. These reactions (RH + HgX2 -- RHgX + Calf-thymus DNA was obtained from Sigma (Grade V), HX) proceed by way of electrophilic substitution and occur while poly(rA) . oligo(dT) and oligo(dT)6 were procured from at the carbon of greatest electron density. For nucleoside Collaborative Research Corp. Poly[d(A-T) I was prepared compounds, the C-5 of pyrimidines, the C-8 of purines, and according to the method