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We have further characterized the polynucleotide ligase purified from cultures of the human heteroploid line EUE [Spadari, Ciarrocchi and Falaschi, Eur. J. Biochem. 22, 75 (1971)]. The 350-fold purified enzyme gives positive response with four different assays; the rates with the different substrates are different, but the variations are identical to those observed with the purified ligase induced by T4-phage. The enzyme can reconstitute the transforming activity of Bacillus subtilis DNA inactivated by pancreatic DNAase. The human cell enzyme is unable to use a hybrid substrate where an interrupted polydeoxynucleotide is annealed to a polyribonucleotide, whereas in the same conditions the T4 enzyme gives appreciable activity. The partial dependence of the purified enzyme on proteins present in a boiled crude extract, reported in the previous paper, seems due to an aspecific protective effect of the soluble proteins of cell extracts. The enzyme does not show any appreciable sequence specificity in the phosphodiester bond it can form. The purified enzyme can be fractionated into two molecular forms, one having a molecular weight of 190000, the other 95000; fresh extracts of EUE cells contain almost exclusively the high molecular-weight form; ageing of the extract or purification lead to the appearance of the second peak, without variations in the total activity. This could correspond to the conversion of a dimer structure into a monomer. The “dimer” has a pH optimum close to 8.1, whereas the “monomer” has its optimum at 7.5; this explains the bimodal pH curve previously described in the purified enzyme. [ABSTRACT FROM AUTHOR]

A DNA polymerase has been extracted from spinach chloroplasts and purified by chromatography on DEAE-cellulose and hydroxyapatite. A great similarity between the purified chloroplast polymerase and the mammalian mitochondrial DNA polymerase γ was found by several criteria: preference for the synthetic primer-template (dT)12–18 · poly(rA), optimal requirement for Mn2+ (0.1–1.0 mM), KCl (100 mM) and pH (8–9). high relative molecular mass (approximately 105000), resistance to aphidicolin and inhibition by N-ethylmalcimide. Some peculiar features of the chloroplast DNA polymerase have, however, been noticed. The mammalian DNA polymerase γ has been suggested to be responsible for the replication of mitochondrial DNA. Thus, both the presence of a γ-like DNA polymerase in chloroplasts and the similarities between the chloroplast and the mitochondrial DNA (absence of a nucleosomal structure and presence of displacement loops) lead to the suggestion that chloroplast DNA is also replicated by a γ-like DNA polymerase and that the γ polymerases present in eukaryotes are, therefore, involved in a strand-displacement DNA synthesis. An α-like DNA polymerase activity, present and predominant in crude leaf extracts, was practically absent from purified chloroplast preparations. [ABSTRACT FROM AUTHOR]

DNA polymerase β was isolated from rat cortex neurons and characterised. Its properties were strikingly similar to those of other mammalian β-polymerases. In adult rats, this was the major DNA polymerase occurring in neuronal nuclei, which contained no α-polymerase, 99.2% β-polymerase and only 0.8% γ-polymerase. Isolated neuronal nuclei of this developmental stage were shown to perform ultraviolet-induced repair DNA synthesis in vitro. Since β-polymerase was virtually the exclusive DNA polymerase in these nuclei it was concluded that the β enzyme was responsible for the observed DNA repair. This was further substantiated by demonstrating a virtually complete suppression of DNA repair in irradiated nuclei by 2′,3′-dideoxyribosylthymine 5′-triphosphate (d2TTP), a potent β-polymerase inhibitor. However, the presence of minute amounts of γ-polymerase in neuronal nuclei and its susceptibility to d2TTP did not allow one to rule out an ancillary role of DNA polymerase γ in DNA repair. In view of the similarity of the neuronal DNA polymerase β with all other mammalian β-polymerases it may be speculated that the ability to perform repair DNA synthesis is not unique to the neuronal enzyme but is a general function of all β-polymerases. [ABSTRACT FROM AUTHOR]

DNA polymerase γ and mitochondrial DNA polymerase were isolated from brain nuclei and synaptosomes respectively. The presence of a single DNA polymerase in synaptosomal mitochondria was established by chromatography on DEAE-cellulose, phosphocellulose and DNA-cellulose, as well as by sedimentation analysis and isoelectric focusing. A great similarity between the purified nuclear DNA polymerase γ and the mitochondrial enzyme was found by the following criteria: chromatographic behaviour in three column systems: essentially complete inhibition by N-ethyl-maleimide (2 mM); optimal requirements for Mn2- (0.1 mM), Mg2+ (5 mM) and pH (8.0): template preferences, poly(A) - (dT)20-25 > activated DNA > poly(dA)-(dT)12-18; lack of activity on single-stranded polynucleotides and (dT)12-primed mRNA : molecular weight (180000), sedimentation (9.2 S) and isoelectric point (pl 5,4). We therefore conclude that brain nuclear DNA polymerase γ and synaptosomal mitochondrial DNA polymerase are closely related and may even be identical. [ABSTRACT FROM AUTHOR]

We have fractionated from human aneuploid cell cultures three different enzyme fractions degrading single-stranded DNA. We have purified and characterized one of these DNases: this is an endonuclease working at alkaline pH (around 9.5) and requiring Mg2+ for its activity. The enzyme degrades denatured DNA over 100 times more efficiently than native DNA in optimal conditions. The termini produced by the enzyme have 5′P and 3′OH ends. The enzyme can attack, though at reduced rate, the supertwisted circular molecule of Simian virus 40 DNA. whereas it is inactive on the nicked circular molecule. The ultraviolet irradiation of DNA, whether native or denatured. does not affect its efficiency as substrate of the DNase. The properties of this endonuclease distinguish it from those of the other DNases described previously in mammalian cells: the denomination DNase VI is therefore proposed. Its properties are similar to those of DNases described in Ustilago maydis and Bacillus subtilis, for which an essential role in recombination seems likely. [ABSTRACT FROM AUTHOR]

A polynucleotide ligase has been detected and purified over 300-fold from human cells cultured in vitro. The enzymic activity is dependent on ATP and Mg2+. The pH curve is bimodal with two optima at pH 7.5 and 8.1. The purified enzyme requires for its activity a heat-resistant protein factor present in the crude extract. [ABSTRACT FROM AUTHOR]

A procedure for the isolation of a pure RNA :DNA hybrid is described. The isolation was accomplished through the following steps: 1. 32P-labeled ribosomal RNA of Bacillus subtilis was hybridized with ³H-labeled DNA of the same organism; 2. the excess of unhybridized DNA was partially hydrolyzed with exonuclease I from Escherichia coli; 3. the mixture was purified by gel-filtration on a Sepharose 4B column, thus eliminating the products of hydrolysis and a considerable fraction of residual unhybridized DNA; 4. filtration through a nitrocellulose column freed the mixture, of another portion of residual denatured DNA without loss of hybrid; 5. two successive CsCl gradients allowed the isolation of material having a DNA/RNA ratio very close to 1, to be compared to an original value of 250 at the beginning of the purification. The overall yield, in different runs, was of approximately 5%. The isolated hybrid does not adsorb to nitrocellulose filters, as expected for its double-stranded structure. The densities of the pure hybrid in CsCl and Cs2SO4 are, respectively, of 1.788 and 1.480 g/cm³. The molecular weight of the DNA moiety of the isolated hybrid is of approximately 50000. The procedure is highly reproducible and can be applied in principle to any hybrid for which a pure RNA is available. [ABSTRACT FROM AUTHOR]

The genes for rRNA of Bacillius subtilis have been purified as high molecular weight DNA: RNA hybrids with a DNA/RNA ratio of 1. Two different methods were used. (1) High molecular weight denatured DNA (Mr = 2.2 × 107) was hybridized with rRNA and the excess of single- stranded DNA was digested with Neurospora crassa endonuclease and Escherichia coli exonuclease I. The hybrid was then purified on Sepharose 4B and two subsequent CsCl gradients. This is a marked improvement on the previously published procedure of Sgaramella, Spadari and Falaschi (1968) insofar as it gives high molecular weight products (8 × 105 for the DNA moiety of the hybrid). (2) A simplified non-enzymic procedure that used relatively short single-stranded DNA molecules (Mr = 4 × 106) and several subsequent CsCl gradients led to a hybrid with a DNA : RNA ratio of 1.1 and a molecular weight higher than the previous procedure (1.7 × 106 for the DNA moiety). The DNA of the isolated hybrid, after removal of the RNA moiety, rehybridizes completely with rRNA. After hybridization of rRNA with DNA having a single-stranded molecular weight of 2.2 × 107, the hybrid bands mainly at a density corresponding to that of denatured DNA; hybrid with & density corresponding to that of 1:1 DNA/RNA ratio can be obtained only if the DNA has been reduced by enzymic digestion or shearing to a single-stranded molecular weight close to 2 × 106; this indicates that between each set of 16-S + 23-S rRNA genes, there must be regions of DNA non-complementary to rRNA; these regions have a single-stranded molecular weight that can be estimated as greater than 1.8x 107. [ABSTRACT FROM AUTHOR]

Focuses on a study which investigated the interaction of human DNA polymerase λ with proliferating cell nuclear antigen (PCNA). Experiments used to demonstrate that DNA polymerase λ interacts with PCNA in vivo in human cells; Importance of residues located in a helix-hairpin-helix motif for the interaction of DNA polymerase λ with primer/template DNA; Schematic diagram of the interaction of p21 with PCNA.

Recently, β-L-nucleoside analogues have emerged as a new class of sugar modified nucleosides with potential antiviral and/or antitumoral activity. As a part of our ongoing research on this topic, we decided to synthesize 5-CF3-β-L-dUrd (7), the hitherto unknown L-enantiomer of Trifluridine, an antiherpetic drug approved by FDA but only used in topical applications due to concomitant cytotoxicity. 5-CF3-β-L-dUrd (7) as well as some other related L-nucleoside derivatives were stereospecifically prepared and tested in vitro against viral (HSV-1 and HSV-2) and human thymidine kinases (TK). [ABSTRACT FROM AUTHOR]