The synthesis and enzymatic polymerization of nucleotides containing mercury: potential tools for nucleic acid sequencing and structural analysis.
نویسندگان
چکیده
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.
منابع مشابه
A Novel Initiator of [5-(benzyloxy)-4-oxo-4H-pyran-2-yl]methyl-2-bromo-2-methylpropanoateas in Atom Transfer Radical Polymerization of Styrene and Methyl Methacrylate
A novel nano-initiator containing kojic acid moiety, [5-(benzyloxy)-4-oxo-4H-pyran-2-yl)methyl-2-bromo-2-methylpropanoate was synthesized by the reaction of 5-(benzyloxy)-2-(hydroxymethyl)-4H-pyran-4-one with 2-bromoisobutyryl bromide in triethylamine and used as initiator in the atom transfer radical polymerization (ATRP) of styrene and methyl methacrylate in the presence of Cu(0)/CuCl2and N,N...
متن کاملCloning and sequencing of rainbow trout (Oncorhynchus mykiss) interferon regulatory factor 7
Interferon regulatory factor 7 (IRF7) gene was cloned from a subtractive cDNA library constructed with mRNAs obtained from rainbow trout (Oncorhynchus mykiss) macrophage cell line (RTS-11). Using expressed sequence tag clones of submitted IRF7 amino acid sequences, specific primers were designed. Results showed that IRF7 cDNA contains an ORF of 1251 nucleotides that translates into a 416 resi...
متن کاملSurface Recognition and Complexations Between Synthetic Poly(ribo)nucleotides and Neutral Phospholipids and Their Implications in Lipofection
Thermodynamic features related to preparation and use of self-assemblies formed between multilamellar and unilamellar zwitterionic liposomes and polynucleotides with various conformation and sizes are presented. The divalent metal cation or surfactant-induced adsorption, aggregation and adhesion between single- and double-stranded polyribonucleotides and phosphatidylcholine vesicles was followe...
متن کاملSurface Recognition and Complexations Between Synthetic Poly(ribo)nucleotides and Neutral Phospholipids and Their Implications in Lipofection
Thermodynamic features related to preparation and use of self-assemblies formed between multilamellar and unilamellar zwitterionic liposomes and polynucleotides with various conformation and sizes are presented. The divalent metal cation or surfactant-induced adsorption, aggregation and adhesion between single- and double-stranded polyribonucleotides and phosphatidylcholine vesicles was followe...
متن کاملSlide into action: dynamic shuttling of HIV reverse transcriptase on nucleic acid substrates.
The reverse transcriptase (RT) of human immunodeficiency virus (HIV) catalyzes a series of reactions to convert single-stranded viral RNA into double-stranded DNA for host cell integration. This process requires a variety of enzymatic activities, including DNA polymerization, RNA cleavage, strand transfer, and strand displacement synthesis. We used single-molecule fluorescence resonance energy ...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- Proceedings of the National Academy of Sciences of the United States of America
دوره 70 8 شماره
صفحات -
تاریخ انتشار 1973