Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1973 Aug;70(8):2238–2242. doi: 10.1073/pnas.70.8.2238

The Synthesis and Enzymatic Polymerization of Nucleotides Containing Mercury: Potential Tools for Nucleic Acid Sequencing and Structural Analysis

R M K Dale 1, D C Livingston 1,*, D C Ward 1
PMCID: PMC433709  PMID: 4365367

Abstract

A simple acetoxymercuration reaction for introducing covalently bound mercury atoms into nucleotides is described. The 5-mercuriacetate derivatives of UTP, CTP, dUTP, and dCTP, as well as the 7-mercuriacetate derivative of 7-deazaATP, have been prepared by this procedure and tested as substrates for nucleic acid polymerases. These nucleotides, in the absence of added mercaptan, are not polymerized and in most instances are potent enzyme inhibitors. However, conversion of these mercuriacetates to mercurithio compounds in situ by the addition of one of various mercaptans, yields nucleoside triphosphates that are excellent substrates for all polymerases tested: Escherichia coli and T7 RNA polymerases, DNA polymerase I of E. coli, DNA polymerase of avian myeloblastosis virus, and calf-thymus terminal deoxynucleotidyl transferase. By varying the mercaptan used to promote syntheses it is possible to access certain structural limitations in the enzyme's nucleoside triphosphate binding site. These mercurinucleotides appear to have a diversity of potential applications: (1) as heavy-atom reagents for crystallographic and microscopic studies; (2) as affinity probes for enzymes sensitive to sulfhydryl modification; (3) as steric probes of substrate-binding sites on enzymes; and (4) as reagents for forming covalent protein-polynucleotide complexes.

Keywords: acetoxymercuration, mercaptans, Escherichia coli, polymerases

Full text

PDF
2238

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Burgess R. R. Separation and characterization of the subunits of ribonucleic acid polymerase. J Biol Chem. 1969 Nov 25;244(22):6168–6176. [PubMed] [Google Scholar]
  2. Crewe A. V., Wall J., Langmore J. Visibility of single atoms. Science. 1970 Jun 12;168(3937):1338–1340. doi: 10.1126/science.168.3937.1338. [DOI] [PubMed] [Google Scholar]
  3. DONOHUE J., TRUEBLOOD K. N. Base pairing in DNA. J Mol Biol. 1960 Dec;2:363–371. doi: 10.1016/s0022-2836(60)80047-2. [DOI] [PubMed] [Google Scholar]
  4. Di Giamberardino L., Koller T., Beer M. Electron microscopic study of the base sequence in nucleic acids. IX. Absence of fragmentation and of cross-linking during reaction with osmium tetroxide and cyanide. Biochim Biophys Acta. 1969 Jun 17;182(2):523–529. [PubMed] [Google Scholar]
  5. Green M., Rokutanda M., Fujinaga K., Ray R. K., Rokutanda H., Gurgo C. Mechanism of carcinogenesis by RNA tumor viruses. I. An RNA-dependent DNA polymerase in murine sarcoma viruses. Proc Natl Acad Sci U S A. 1970 Sep;67(1):385–393. doi: 10.1073/pnas.67.1.385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Jovin T. M., Englund P. T., Bertsch L. L. Enzymatic synthesis of deoxyribonucleic acid. XXVI. Physical and chemical studies of a homogeneous deoxyribonucleic acid polymerase. J Biol Chem. 1969 Jun 10;244(11):2996–3008. [PubMed] [Google Scholar]
  7. Kapuler A. M., Spiegelman S. Q-beta-replicase and E. coli transcriptase: requirements for substrate selection as revealed by a study of base analogs. Proc Natl Acad Sci U S A. 1970 Jun;66(2):539–546. doi: 10.1073/pnas.66.2.539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kapuler A. M., Ward D. C., Mendelsohn N., Klett H., Acs G. Utilization of substrate analogs by mengovirus induced RNA polymerase. Virology. 1969 Apr;37(4):701–706. doi: 10.1016/0042-6822(69)90295-5. [DOI] [PubMed] [Google Scholar]
  9. MOUDRIANAKIS E. N., BEER M. BASE SEQUENCE DETERMINATION IN NUCLEIC ACIDS WITH THE ELECTRON MICROSCOPE. 3. CHEMISTRY AND MICROSCOPY OF GUANINE-LABELED DNA. Proc Natl Acad Sci U S A. 1965 Mar;53:564–571. doi: 10.1073/pnas.53.3.564. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Michelson A. M., Monny C., Kapuler A. M. Poly 8-bromoguanylic acid. Biochim Biophys Acta. 1970 Sep 17;217(1):7–17. doi: 10.1016/0005-2787(70)90117-6. [DOI] [PubMed] [Google Scholar]
  11. Ottensmeyer F. P., Schmidt E. E., Olbrecht A. J. Image of a sulfur atom. Science. 1973 Jan 12;179(4069):175–176. doi: 10.1126/science.179.4069.175. [DOI] [PubMed] [Google Scholar]
  12. Pietrzykowska I., Shugar D. Studies on bacteriophage and bacteriophage DNA containing 5-ethyluracil or 5-bromouracil in place of thymine. Acta Biochim Pol. 1967;14(1):169–181. [PubMed] [Google Scholar]
  13. Pullman B., Pullman A. Quantum-mechanical investigations of the electronic structure of nucleic acids and their constituents. Prog Nucleic Acid Res Mol Biol. 1969;9:327–402. doi: 10.1016/s0079-6603(08)60772-2. [DOI] [PubMed] [Google Scholar]
  14. RICHARDSON C. C., LEHMAN I. R., KORNBERG A. A DEOXYRIBONUCLEIC ACID PHOSPHATASE-EXONUCLEASE FROM ESCHERICHIA COLI. II. CHARACTERIZATION OF THE EXONUCLEASE ACTIVITY. J Biol Chem. 1964 Jan;239:251–258. [PubMed] [Google Scholar]
  15. Roy-Burman S., Roy-Burman P., Visser D. W. Inhibition of ribonucleic acid polymerase by 5-hydroxyuridine 5'-triphosphate. J Biol Chem. 1966 Feb 25;241(4):781–786. [PubMed] [Google Scholar]
  16. Tolman R. L., Robins R. K., Townsend L. B. Pyrrolopyrimidine nucleosides. 3. The total synthesis of toyocamycin, sangivamycin, tubercidin, and related derivatives. J Am Chem Soc. 1969 Apr 9;91(8):2102–2108. doi: 10.1021/ja01036a040. [DOI] [PubMed] [Google Scholar]
  17. Ts'o P. O., Kondo N. S., Schweizer M. P., Hollis D. P. Studies of the conformation and interaction in dinucleoside mono- and diphosphates by proton magnetic resonance. Biochemistry. 1969 Mar;8(3):997–1029. doi: 10.1021/bi00831a033. [DOI] [PubMed] [Google Scholar]
  18. Ward D. C., Cerami A., Reich E., Acs G., Altwerger L. Biochemical studies of the nucleoside analogue, formycin. J Biol Chem. 1969 Jun 25;244(12):3243–3250. [PubMed] [Google Scholar]
  19. Whiting R. F., Ottensmeyer F. P. Heavy atoms in model compounds and nucleic acid imaged by dark field transmission electron microscopy. J Mol Biol. 1972 Jun 20;67(2):173–181. doi: 10.1016/0022-2836(72)90234-3. [DOI] [PubMed] [Google Scholar]
  20. YONEDA M., BOLLUM F. J. DEOXYNUCLEOTIDE-POLYMERIZING ENZYMES OF CALF THYMUS GLAND. I. LARGE SCALE PURIFICATION OF TERMINAL AND REPLICATIVE DEOXYNUCLEOTIDYL TRANSFERASES. J Biol Chem. 1965 Aug;240:3385–3391. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES