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Enzymatic Synthesis of Deoxyribonucleotides

1963; Elsevier BV; Volume: 238; Issue: 10 Linguagem: Inglês

10.1016/s0021-9258(18)48681-4

1083-351X

L. Elizabeth Bertani, Agneta Häggmark, Peter Reichard,

Chemical Synthesis and Analysis

The purification and properties of an enzyme system ("Fraction B") isolated from Escherichia coli B were recently described (1,2).It was shown that Fraction B in the presence of adenosine triphosphate, Mg", and reduced lipoate catalyzed the formation of deoxycytidine diphosphate from cytidine diphosphate and also the formation of deoxyuridine phosphate derivatives from uridine diphosphate (UDP).In addition, Fraction B was found to contain a specific pyrophosphatase that cleaved dUTP to dUMP and pyrophosphate.It was suggested that this latter enzyme might account for the absence of uracil in deoxyribonucleic acid.On the basis of these reactions, and others not previously reported in detail, it was postulated that the formation and transformations of deoxyuridine phosphates occurred according to the following scheme. UMP + UDP --+ dUDP F? dUTP + dUMP + pyrophosphsteThe present paper presents more fully the experiments which support this postulated reaction sequence.The existence of a dUTP pyrophosphatase in E. coli has been independently discovered by Greenberg and Somerville (3). EXPERIMENTAL PROCEDURE MatiEs and MethodaNonlabeled ribo-and deoxyribonucleotides were obtained from Sigma Chemical Company and from California Corporation for Biochemical Research.The latter company also supplied 2-Cl'-uracil.Ha-Labeled dCMP was obtained from Schwarz Bio-Research, Inc. dUDP and dUTP were obtained by deamination of the commercially available cytosine compounds (4).Unless otherwise stated, all paper chromatograms (descending technique) were run at 25 i lo.The separation of the mono-, di-, and triphosphates of either uridine or deoxyuridine was carried out with an isobutyric acid-ammonia system (5).Uridine and deoxyuridine compounds were separated with an ammonium acetateethanol-borate system (6, 7).The uracilcontaining compounds were localized on the dried papers with the aid of a Mineral&e lamp.