The synthesis and enzymatic polymerization of nucleotides containing mercury: potential tools for nucleic acid sequencing and structural analysis.
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.
D000227:Adenine Nucleotides; D000255:Adenosine Triphosphate / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D001354:Avian Leukosis Virus / Q000201:enzymology; D002417:Cattle; D003597:Cytosine Nucleotides / Q000138:chemical synthesis / Q000378:metabolism; D004254:DNA Nucleotidyltransferases / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006160:Guanosine Triphosphate / Q000378:metabolism; D008628:Mercury; D008629:Mercury Isotopes; D009711:Nucleotides / Q000138:chemical synthesis* / Q000378:metabolism; D009942:Organometallic Compounds / Q000138:chemical synthesis* / Q000378:metabolism; D011119:Polynucleotides / Q000096:biosynthesis; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013698:Templates, Genetic; D013942:Thymine Nucleotides; D013950:Thymus Gland / Q000201:enzymology; D014316:Tritium; D014500:Uracil Nucleotides / Q000138:chemical synthesis
D000227:Adenine Nucleotides; D003597:Cytosine Nucleotides; D008629:Mercury Isotopes; D009711:Nucleotides; D009942:Organometallic Compounds; D011119:Polynucleotides; D013438:Sulfhydryl Compounds; D013942:Thymine Nucleotides; D014500:Uracil Nucleotides; D014316:Tritium; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004254:DNA Nucleotidyltransferases; D012321:DNA-Directed RNA Polymerases; D008628:Mercury