Polynucleotide synthetase of E. coli: an enzyme complex having polynucleotide phosphorylase as apoenzyme.

A previously described synthetase system of Escherichia coli that utilizes ribonucleoside triphosphates has been purified extensively and shown to consist of an apoenzyme and three protein factors. The apoenzyme itself was revealed to be polynucleotide phosphorylase. The conditions under which the latter - an enzyme incorporating nucleoside diphosphates - is converted to a system catalyzing the uptake of nucleoside triphosphates have been studied in detail with respect to primer requirements, the influence of triphosphates on diphosphate utilization and vice versa, and the possibly regulatory effect of the guanosine di- and triphosphates. The fully supplemented enzyme system (polynucleotide synthetase) incorporates GTP only in the presence of ATP, producing a polynucleotide with an A : G ratio near unity.

Simplified method for purification of ribonuclease H from calf thymus.

An improved purification procedure for the isolation of ribonuclease H(hybrid nuclease; RNA-DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) from the thymus of 4-to 6-months-old calves yields two highly active forms of the enzyme, designated as ribonuclease H1 and H2. Their isoelectric points are 5.0 +/- 0.05 and 5.25 +/- 0.05, respectively; their molecular weight, estimated from gel filtration, is in both cases 64,000 +/- 2000. On sodium dodecyl sulfate gel electrophoresis, two principal bands were identified, with molecular weights of 32,000 and 21,000. The nature of the nucleotides at the 3'-OH terminals, produced initially by the enzymic hydrolysis of hybridized RNA, was examined and shown to be a function of the divalent metal ion employed as activator.

Action of nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and nicotinamide mononucleotide.

The action of the nucleotide phosphotransferase of Escherichia coli on nicotinamide riboside and on its 5'-phosphate results in the addition of one phosphate moiety to each of the substrates. Although the proof is not conclusive, it is likely that the phosphate group is transferred to the 3'-hydroxyl of the ribose. This is in contrast to the behavior of the enzyme toward NAD in which only the adenylic acid portion is phosphorylated enzymically.

Nucleotide phosphotransferase of Escherichia coli: purification by affinity chromatography.

Improved extraction and purification procedures permit the isolation from Escherichia coli W cells of much larger quantities and of more highly purified preparations of nucleotide phosphotransferase. Of various affinity resins tested for efficiency of purification, columns of agarose/5'-AMP (AGAMP), type 3, proved the best. In this way a 300- to 450-fold purification of the enzyme was achieved in a few steps. The enzyme, which, as reported before, transfers organically bound phosphate to the 2' or 3' hydroxyls of nucleosides and nucleotides, was tested in its behavior toward a series of ribonucleosidonucleotides, namely, CpC, ApA, CpA, and ApC. All were phosphate acceptors, but a detailed comparative study of adenosine and cytidine, 5'-AMP and 5'-CMP, and ApA and ApC revealed peculiar specificities in the relative distribution of the phosphorylated products.

An assay of ribonuclease H, endoribonucleases, and phosphatases.

An assay for ribonuclease H (EC 3.1.4.34) is described which permits the estimation of the number and the type of cleavages produced by the enzyme when acting on the ribo moiety of a DNA-RNA hybrid substrate. With six different homopolymer hybrids tested, the number of enzymically induced breaks varied widely. The method is applicable to other endoribonucleases, phosphodiesterases, and phosphatases.

Ribonuclease H of calf thymus: substrate specificity, activation, inhibition.

When the action of highly purified specimens of ribonuclease H (hybrid nuclease; RNA-DNA hybrid ribonucleotidohydrolase; EC 3.1.4.34) of calf thymus on a wide selection of homopolymer hybrids was studied, the extent, and even the occurrence, of hydrolysis was found to be governed by the interplay of several factors: the composition of the ribo strand, the length of the deoxyribo strand, and the nature of the activating metal. Mn2+ activates the enzymic cleavage of all hybrid combinations, Mg2+ only of those containing purine ribo strands, Co2+ only of poly(A) hybrids. A 1:1 hybrid of phage f1 DNA and RNA is, however, split in the presence of any of these activators. Hybrids with deoxyribo tetranucleotides can still be cleaved, but not with dinucleotides. The behavior of hybrids containing covalently linked runs of ribo and deoxyribopolynucleotides was studied with the hybrid poly(dT)-poly(A)7-(dA)X]. This hybrid is attacked by ribonuclease H so that the bulk of the resulting poly(dA) still retains one covalently linked riboadenylic acid end group, whereas a small proportion carries a ribo dinucleotide. Inhibition studies showed that ribonuclease H is inactivated irreversibly by pretreatment with S-adenosylmethionine at 35 degrees, but not at 0 degrees. S-Adenosylhomocysteine also is inhibitory, but not irreversibly; also it is essentially limited to the inhibition of the cleavage of purine ribo strands. When the enzyme is exposed simultaneously to both inhibitors, irreversible inactivation is diminished considerably.

A DNA polymerase from embryos of Drosophila melanogaster. Purification and properties.

The more than 2,300-fold purification of a DNA polymerase from the embryos of Drosophila melanogaster is described. The enzyme, which forms a single band on gel electrophoresis, has a molecular weight of about 87,000 and a pH optimum of 8.5. A divalent metal is required for activity, Mg2+ being preferred with activated DNA, Mn2+ with homopolymer template-primers. The enzyme is inactivated completely by mercurials; polyamines are also inhibitory with certain templates. The most efficient template-primer is activated DNA, but homopolymers such as poly(dA)-oligo(dT), poly(A)-oligo(dT), and poly(A)-oligo(U) are also utilized with high efficiency. The purified enzyme preparations appear to be devoid of nuclease activity when assayed directly with suitable substrates. In addition, neither primer nor product is degraded after prolonged incubation with the enzyme. In accordance with previous observations on other DNA polymerases, the Drosophila enzyme can replicate single-stranded DNA only under conditions of simultaneous transcription by RNA polymerase.