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The kinetics of the phosphorolysis of polynucleotide (as differentiated from oligonucleotide) by polynucleotide phosphorylase of Micrococcus luteus has been investigated. Double reciprocal plots of initial velocity against either inorganic phosphate or polynucleotide concentration are linear, and furthermore, the affinity of the enzyme for either substrate is unaffected by the presence of the other. dADP, an analogue of ADP product, is a competitive inhibitor with respect to Pi and polynucleotidy. (Ap)tA-cyclic-p is a competitive inhibitor with respect to Pi. The results are almost identical with both primer-independent (Form-I) and primer-dependent (Form-T) enzymes, although the various kinetic constants differ. On the vasis of these data a rapid equilibrium random Bi Bi mechanism is proposed. The demonstration of two different inhibitor constants for dADP and the difference between the Michaelis and the inhibitor constant for polyadenylic acid in polynucleotide phosphorolysis indicate at least two binding sites for polyadenylic acid and dADP on M. luteus polynucleotide phosphorylase. Its is suggested that in the phosphorolysis of long chain polymers the second binding site permits the polynucleotide to snap right back into position after removal of I mononucleotide unit and thus leads to the observed processive degradation. A general discussion of oligonucleotide and polynucleotide phosphorolysis and the differences between Form-I and Form-T enzymes in de novo synthesis and degradation of polynucleotides is presented.
DNA formed an insoluble complex on mixing with chitosan (poly-D-glucosamine) in solution. The DNA content of the complex was about 50% and the DNA remained insoluble in aqueous media of pH 2-7%; e.g., on treatment of the DNA-chitosan complex with phosphate-buffered saline at pH 7 and 37 degrees C for 26 h, the DNA released into the aqueous phase was less than 0.05%. Obviously, DNA and chitosan formed a tight complex due to ionic interactions. The DNA can be solubilized by treatment with 0.1 N NaOH. RNA and other polynucleotides formed similar insoluble complexes with chitosan. The DNA attached to chitosan can be digested with a mixture of DNase I and phosphodiesterase. Cytosine residues in the DNA (denatured DNA) can be deaminated by treatment with sodium bisulfite, forming uracil DNA-chitosan. The uracil DNA-chitosan served as a substrate for uracil DNA glycosylase. Using polynucleotide-chitosan as an adsorbent, the affinities of reagents for polynucleotides can be determined directly. With this technique it was found that carcinogenic heterocyclic amines have an affinity for RNA as well as DNA. The results with homopolyribonucleotide-chitosans as adsorbents for 4 heterocyclic amines indicated that the binding occurs in a purine nucleotide-specific manner. These results suggest that the polynucleotides in the chitosan complex are accessible to enzymes and reagents. This new derivative may be useful in chemical and biological studies of polynucleotides and substances interacting with polynucleotides.
Poly(formycin phosphate) and poly(laurusin phosphate) were synthesized by polymerizing formycin and laurusin 5'-diphosphate by means of E. coli polynucleotide phosphorylase. The complex formation of these polynucleotides with cyclonucleoside polynucleotides were investigated. While poly(formycin phosphate) did not form the complex with an octanucleotide of 6,2'-anhydro-6-oxy-1-beta-D-arabinofuranosyluracil, poly(laurusin phosphate) did form a 1: 1 complex with octanucleotide of 8,2'-anhydro-8-mercapto-9-beta-D-arabinofuranosyladenine in the presence of 0.15M Na ion at neutrality and 3(o). CD spectrum of this complex showed a couple of a trough at 286 nm and a peak at 262 nm. This fact suggests that the complex has a left-handed helical conformation, which is opposite to the natural double helical polynucleotides. The cause of this phenomenon was discussed in connection with the complex of cyclonucleoside oligonucleotides.
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.
It is already known that modification of E. coli polynucleotide phosphorylase by endogenous proteolysis induces drastic changes in both phosphorolysis and polymerisation reactions. The structural parameters of the proteolysed polynucleotide phosphorylase are described. The phosphorolysis of polynucleotide, which is quite progressive for the native enzyme, is shown to be only partially progressive for the degraded enzyme, owing to the loss of polymer attachment sites.
Schizophyllan is an extracellular polysaccharide consisting of a beta-1,3-D-glucan main chain and exists as a triple helix in water and as a single chain in dimethyl sulfoxide (DMSO). When the single chain of schizophyllan (s-SPG) was mixed with poly(C), poly(A), poly(dA), or poly(dT), they form a macromolecular complex. On the other hand, poly(G), poly(U), poly(I), poly(dG), and poly(dC) do not. This nucleotide specificity evidences that the hydrogen bonds are essential to form the complex, because the former nucleotides have an unoccupied hydrogen-bonding site and the latter ones use the hydrogen-bonding sites in the intramolecular aggregation (i.e., such as the G quartet for poly(G) and poly(dG) and the U hairpin for poly(U)). The hypochromic effect and the increment in the circular dichroism (CD) intensity are observed in accordance with the complex formation. These facts indicate that the base stacking is enhanced in the complex. The solvent-composition (DMSO/water) dependence demonstrates that the hydrophobic interaction is important to form the complex as well as the hydrogen-bonding interaction. With increasing temperature the complex dissociates cooperatively and the melting curve enables the thermodynamic parameters to be evaluated (delta H = -60 to 70 kcal mol-1 and delta S = -150 to 200 cal mol-1 K-1). These values are comparable with those for double helix DNA. Namely, the complex can be characterized by enhancement of the base stacking, cooperative dissociation, the similar thermodynamic parameters to DNA, and combination of the hydrogen-bonding and hydrophobic interactions to form the higher-order structure. These facts surprisingly coincide with characters of the double helix of DNA. In other words, the s-SPG molecule behaves as if it were a complementary polynucleotide chain for the corresponding polynucleotide. Furthermore, stoichiometric study suggested that the complex structure is a triple helix consisting of two s-SPG and one poly(C) or poly(A) chains.
A polymerase activity is associated with protein IV, a protein which is associated with the DNA in bacteriophage PM2. The native enzyme unit is probably a dimer. Manganese ions are required for the polymerisation reaction and there is a well-defined Mn2+ optimum at 2.5 mM. The pH optimum is at 8.1, the temperature optimum at 28 degrees C. The activity is a polynucleotide-pyrophosphorylating reaction in the presence of ribo- or deoxyribonucleoside triphosphates. The polymerisation reaction is stimulated in the presence of nuclei- acids or polynucleotides as effectors. The product is not covalently linked to the effector.
Messenger activity of synthetic tri- and polynucleotide analogs was studied by binding of 14C-labeled aminoacyl-tRNAs to ribosomes in the presence of the analogs. Synthetic messengers used were: poly(A) analogs in which adenosine was replaced by tubercidine (I), 3-deazaadenosine (II), 1-deazaadenosine (III) and 2-methyladenosine (IV); copolymers of adenosine and aristeromycin (V); cyclic triadenylate (VI); the heptanucleotide of 6,2'-O-cyclouridine (VII); the pentanucleotide of 8,2'-S-cycloadenosine (VIIIa); A-U-G analogs in which adenosine was replaced by 8,2'-O- and S-cycloadenosine (VIII), 8,5'-O- and S-cycloadenosine (IX); 8-oxyadenosine (x); 8-bromoadenosine (XI) and formycine (XII). Among these oligo- and polynucleotides, analogs which contained nucleotides of anti conformation having appropriate bases for Watson-Crick type hydrogen bonding stimulated the binding of corresponding tRNAs to ribosomes.
Osmium tetroxide in the presence of pyridine or 2,2'-bipyridine has been found to react completely with the pyrimidine moieties (thymine, uracil, and cytosine) in polynucleotides. Pyrimidine osmate ester moieties, L2OSO4-pyrimidine, were formed. The OSO4 has added across the 5,6 double bond and L=pyridine or 1/2-bipyridine. The pyridine derivatives were not stable and decomposed slowly after the OSO4-pyridine reagent was removed by gel chromatography. Labeled poly(uridylic acid) lost osmium completely during gel chromatography unless the eluent contained a high concentration of pyridine. The products formed between OSO4-bipyridine and polynucleotides were much more stable and the OS label was retained during and after gel chromatography. Both the OSO4-pyridine and OSO4-bipyridine reagents reacted more rapidly than the OSO4-CN-reagent.
The polynucleotide ligase isolated from T4-infected Escherichia coli was previously shown to bring about repair of breaks in the single strands of bihelical DNA. The present work shows that the enzyme can also catalyze the joining of DNA duplexes at their base-paired ends. This novel reaction occurs when the deoxynucleoside at a 5'-end carries a phosphate group and the complementary deoxynucleoside opposite to it carries a 3'-hydroxyl group. The consequence is the lengthening of the original duplex to form dimers or oligomers depending upon whether one or both ends are base-paired.
Mammalian polynucleotide kinases (PNKs) carry out 5'-phosphorylation of nucleic acids. Although the cellular function(s) of these enzymes remain to be delineated, important suggestions have included a role in DNA repair and, more recently, in DNA replication. Like T4 PNK, some preparations of mammalian PNKs have been reported to have an associated 3'-phosphatase activity. Previously, we have identified in calf thymus glands an apparently novel PNK with a neutral to alkaline pH optimum that lacked 3'-phosphatase activity. In this report, we describe purification of another bovine PNK, SNQI-PNK, with a slightly acidic pH optimum that copurifies with a 3'-phosphatase activity. The enzyme appears to be a monomer of 60 kDa. Mammalian DNA replication reactions were supplemented with T4 PNK or SNQI-PNK, and no significant effect on DNA replication in vitro was observed. Database searches support the earlier mapping of the 3'-phosphatase activity of T4 PNK to the C-terminus and suggest that the 3'-phosphatase domain of T4 PNK is related to the protein superfamily of L-2-haloacid dehalogenases. Exopeptidase digestion experiments were carried out to compare the SNQI-PNK enzyme with T4 PNK and led to the inference that the domain organization of the bovine polypeptide may differ from that of the T4 enzyme.
The end-healing and end-sealing steps of the phage T4-induced RNA restriction-repair pathway are performed by two separate enzymes, a bifunctional polynucleotide 5'-kinase/3'-phosphatase and an ATP-dependent RNA ligase. Here we show that a single trifunctional baculovirus enzyme, RNA ligase 1 (Rnl1), catalyzes the identical set of RNA repair reactions. Three enzymatic activities of baculovirus Rnl1 are organized in a modular fashion within a 694-amino acid polypeptide consisting of an autonomous N-terminal RNA-specific ligase domain, Rnl1-(1-385), and a C-terminal kinase-phosphatase domain, Rnl1-(394-694). The ligase domain is itself composed of two functional units. The N-terminal module Rnl1-(1-270) contains essential nucleotidyltransferase motifs I, IV, and V and suffices for both enzyme adenylylation (step 1 of the ligation pathway) and phosphodiester bond formation at a preactivated RNA-adenylate end (step 3). The downstream module extending to residue 385 is required for ligation of a phosphorylated RNA substrate, suggesting that it is involved specifically in the second step of the end-joining pathway, the transfer of AMP from the ligase to the 5'-PO(4) end to form RNA-adenylate. The end-healing domain Rnl1-(394-694) consists of a proximal 5'-kinase module with an essential P-loop motif ((404)GSGKS(408)) and a distal 3'-phosphatase module with an essential acylphosphatase motif ((560)DLDGT(564)). Our findings have implications for the evolution of RNA repair systems and their potential roles in virus-host dynamics.
2'-O-o-Nitrobenzyluridine, -cytidine and -adenosine were phosphorylated with phosphoryl chloride to the corresponding 5'-phosphates and led to 5'-diphosphates by the method of Moffatt and Khorana. These 2'-O-oNB-nucleoside 5'-diphosphates were incubated with a primer CpApA and polynucleotide phosphorylase in the presence of Mn2+. Tetranucleotides CpApApU, CpApApC and CpApApA were obtained after photosensitive removal of oNB groups in yields of 54-70%.
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