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PubMed1976

Two membrane sites for DNA synthesis in Pneumococcus.

A DNA membrane fraction extracted from pneumococci can be separated into two subfractions with respect to macromolecular composition and DNA synthesis by centrifugation in a 30-60% w/v neutral sucrose gradient. Each fraction can be rebanded in a sucrose gradient or centrifuged to equilibrium in a CsCl density gradient without altering the ability of the fractions to synthesize DNA. The fast sedimenting (heavy) fraction contains 45% of the DNA, and the bulk of the phospholipid, protein, and RNA. The light fraction contains 50% of the DNA, and lower, but significant amounts of phospholipid, RNA, and protein. Both fractions contain a DNA replication complex consisting of a number of enzymes involved in synthesizing DNA or DNA precursors, as well as RNA polymerase activity. However, the specific activity of DNA polymerase in the light fraction is much greater than that in the heavy fraction. In addition, the following results suggest that the former is concerned primarily with replication of the genome while the latter has characteristics of a repair function for the genome. (1) newly synthesized DNA can be detected within 30 s in the light fraction but not until 4 min in the heavy fraction. (2) an RNA-DNA single-stranded hybrid can be demonstrated during initial stages of DNA synthesis in the light, but not heavy fraction. (3) extensive semiconservative DNA replication occurs in the light fraction, whereas little such replication is detected in the heavy fraction. (4) DNA polymerase activity in the light fraction has several of the characteristics of a polymerase identified by others as being concerned with normal DNA replication, such as inhibition by N-ethylmaleimide, and relatively high rates of chain elongation (4.9 x 10(4) nucleotides/min). In contrast, DNA polymerase activity in the heavy fraction has characteristic properties associated with DNA polymerase I, a possible repair enzyme. These include higher activity for a d(A-T)n template than that detected in the light fraction, no effect of N-ethylmaleimide, and relatively low rates of chain elongation (9 x 10(3) nucleotides/min).

Cell Membrane↗
PubMed1977

Serological analysis of cellular and viral DNA polymerases by an antiserum to DNA polymerase gamma of human lymphoblasts.

An antiserum has been prepared against a highly purified DNA polymerase gamma from NC37 cells, a normal human lymphoblast cell line. The antiserum does not possess enzyme neutralizing activity, but does bind specifically to DNA polymerase gamma. When tested in a double antibody immunoprecipitation assay, the antibody does not cross-react with DNA polymerases alpha or beta, purified from NC37 cells, or with reverse transcriptases of avian, murine, or primate RNA tumor viruses. Antisera prepared against purified reverse transcriptases similarly do not recognize DNA polymerase gamma, either in an enzyme neutralization assay or in the more sensitive double antibody immunoprecipitation assay. The availability of an antiserum to DNA polymerase gamma will allow the further characterization of enzyme activities isolated from cellular material and suspected of being related to viral reverse ttranscriptases. In those cases where such activities do not immunologically resemble known viral DNA polymerases, the anti-DNA polymerase gamma will help determine the viral or cellular nature of the unknown activity.

Antigen-Antibody Reactions↗
PubMed2024

N6-methyladenosine modification of a parvovirus-encoded small noncoding RNA facilitates viral DNA replication through recruiting Y-family DNA polymerases.

Human bocavirus 1 (HBoV1) is a human parvovirus that causes lower respiratory tract infections in young children. It contains a single-stranded (ss) DNA genome of ~5.5 kb that encodes a small noncoding RNA of 140 nucleotides known as bocavirus-encoded small RNA (BocaSR), in addition to viral proteins. Here, we determined the secondary structure of BocaSR in vivo by using DMS-MaPseq. Our findings reveal that BocaSR undergoes N6-methyladenosine (m6A) modification at multiple sites, which is critical for viral DNA replication in both dividing HEK293 cells and nondividing cells of the human airway epithelium. Mechanistically, we found that m6A-modified BocaSR serves as a mediator for recruiting Y-family DNA repair DNA polymerase (Pol) η and Pol κ likely through a direct interaction between BocaSR and the viral DNA replication origin at the right terminus of the viral genome. Thus, this report represents direct involvement of a viral small noncoding RNA in viral DNA replication through m6A modification.

Humans↗
PubMed1976

Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus.

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.

Avian Myeloblastosis Virus↗
PubMed1977

Oligo(dG)12-18 aggregates result in non-homogeneity of oligo(dG)12-18.poly(C) type primer-template.

Studies on the absorption spectra of equimolar solutions of oligo(dG)12-18 and poly(C), poly(Cm) or poly(Ce) showed that only 13%, 3% and 3% of base pairs, repectively, form complexes. Upon centrifugation of oligo(dG)12-18 with a molar excess of poly(C) of poly(Cm) in an analytical and a preparative centrifuge, it was found that only a part of oligodeoxynucleotide sediments with the polynucleotide, i.e. more rapidly than oligo(dG)12-18, poly(C) or poly(Cm). Products of binding of oligo(dG)12-18 with poly(C), poly(Cm) or poly (Ce) direct the synthesis of poly (dG) by AMV reverse transcriptas in accordance with the reported characteristics of these primer-templates, as well as of the enzyme. These observations suggest that the solutions of oligo(dG)12-18 with (polyC) or its analogues, commonly used as primer-templates of RNA- and DNA-directed DNA polymerases, contain a polynucleotide, to which oligo(dG)12-18 aggregates are bound through a few nucleotides chains shorter than 12-18 residues. These chains of oligo(dG)12-18 containing the 3'-OH ends are capable of of initiating the reaction with DNA POLYMERASES.

Avian Myeloblastosis Virus↗
PubMed2026

Refining a giant virus lineage: a novel order unifying Mamonoviridae and "Manesviridae," unveiled by the discovery of furtivovirus.

UNLABELLED: The evolutionary origins and taxonomic framework of giant viruses related to the family Mamonoviridae and its relative group, including clandestinovirus, remain unclassified due to gaps in genome size and host range between these two groups. This study aimed to address this gap by integrating our newly isolated virus with publicly available metagenome-assembled genomes (MAGs) to construct a more robust phylogenetic framework. Here, we report the isolation and characterization of a new giant virus, furtivovirus, using the unicellular amoeba Vermamoeba vermiformis as a host. Furtivovirus has a genome of approximately 560 kbp and shares key features with its closest relative, clandestinovirus. Ultrastructural analysis revealed a unique host-nucleus-dependent replication strategy characterized by the breakdown of the nuclear membrane and the packaging of nascent virions directly within the nucleoplasm, distinguishing it from canonical cytoplasmic virion factories. Comprehensive phylogenetic and comparative genomic analyses of shared orthologous groups and nucleocytovirus marker proteins revealed that furtivovirus, clandestinovirus, ushikuvirus, and usurpativirus form a distinct monophyletic clade, for which we propose a new family, "Manesviridae." Further analysis using amino acid-based similarity metrics of Nucleocytoviricota viral genomes, including established MAGs, demonstrated that this new family is robustly placed as a sister group to the family Mamonoviridae. This study elucidated the evolutionary relationships between viruses with large and small genomes that possess similar virion sizes within this lineage. Based on this cumulative evidence, we propose the establishment of a new order to unify these two families, thereby expanding their diversity and clarifying the evolutionary history of this branch within Nucleocytoviricota. IMPORTANCE: Giant viruses challenge our traditional understanding of viral evolution, raising the question of how a single related group can diverge to infect different hosts while evolving into vastly different genome sizes and replication strategies. The family Mamonoviridae and its relatives epitomize this evolutionary divergence: one group possesses massive genomes, whereas the other has genomes that are less than half their size. The discovery of furtivovirus and its unique nucleoplasm-dependent replication cycle provides a critical biological context for this genomic disparity. Through deep comparative genomic analysis, we demonstrated that these seemingly disparate lineages share a cohesive evolutionary origin that is distinct from other established orders. This finding highlights the complexity of genome evolution, demonstrating that giant viruses can expand their overall genome size to adapt to uncertain environments while reducing their core essential genes, thereby providing new insights into the evolutionary pressures that shape the diversity of the virosphere.

Giant Viruses↗
PubMed1976

DNA polymerases of murine LBN/b leukemic cells.

In the post-mitochondrial fraction of murine LBN/b leukemic cells, four fractions with DNA polymerase activity (I, II, III, IV) were found. On the basis of ion exchanger affinity and poly(A), poly(C) and poly(Cm) replication ability, fraction I was classified as RNA-directed DNA polymerase of viral origin. On the basis of the differences in the ion exchanger affinity, molecular weight, template requirement, pH-dependence of enzymatic activity and NaCl concentration, divalent ion requirements and susceptibility to N-ethylmaleimide inhibition, fractions II, III and IV were classified as DNA-directed DNA polymerases beta, alpha and gamma, respectively. Three fractions, i.e. reverse transcriptase, and DNA-directed DNA polymerases beta and gamma, were found to incorporate dTMP on a poly(A)-oligo(dT) template-primer. Despite the similarity of the reaction of DNA polymerases beta and gamma with poly(A)-oligo(dT), some other properties of these enzymes suggest that they represent distinct enzymatic entities.

Animals↗
PubMed1976

Separation of cellular and viral DNA polymerases by affinity chromatography on polynucleotide-Sepharose.

Polyguanylate- and poly(2'-O-methyl)uridylate-Sepharose have been prepared for affinity chromatography of DNA polymerases of viral origin (reverse transcriptase). Both cellular DNA polymerases and reverse transcriptase bind to polyguanylate-Sepharose. The cellular polymerases can be eluted from the column between 0.32 and 0.42 M NaCl while reverse transcriptase eluted between 0.56 and 0.78 M NaCl. However, only reverse transcriptase adheres to poly(2'-O-methyl)uridylate-Sepharose and can be eluted at approximately 0.35 M NaCl. The columns were used to partially purify RNA-dependent DNA polymerase from spleens of mice infected with Rauscher leukemia virus. The enzyme preparation is about 1300-fold purified and is inhibited by antiserum prepared against purified reverse transcriptase from Rauscher leukemia virus to the same extent as the virion enzyme.

Animals↗
PubMed1976

Absence of thymus-derived lymphocyte markers in myelogenous leukemia (Ph1+) cell line K-562.

The myelogenous leukemia cell line K-562 with a Ph1+chromosome, derived from a patient with chronic myelogenous leukemia in terminal blastic crisis, is not a bone marrow-derived lymphoblastic cell line, because the cells neither produce immunoglobulins nor possess complement receptors. Since it has been suspected that blasts found in some patients with chronic myelogenous leukemia in blastic crisis might be thymus-derived cells, we have studied several parameters to demonstrate that K-562 cells are not thymus-derived lymphoblasts. The results of this study show: (a) no cross-reactivity of antisera to K-562 cells with normal human thymocytes; (b) lack of cytotoxicity of a specific horse anti-human thymocyte globulin for K-562 cells; (c) failure of the treatment of K-562 cells with bovine thymosin to induce antigenic determinant and erythrocyte rosette receptors on K-562 cells; (d) presence of receptors for the Fc portion of immunoglobulin G; (e) absence of terminal deoxynucleotidyl transferase; and (f) cytotoxicity of monkey antiserum to K-562 cells for malignant thymus-derived cells (Molt-4). However, absorption with Molt-4 cells abolished the cross-reactivity with Molt-4 cells, whereas 60% of the antibody to K-562 cells remained in the immune serum. Studies of DNA polymerase activities revealed that K-562 cells have levels of polymerase alpha and beta, like other proliferating cells, and an RNA-dependent DNA polymerase activity, presumably representing polymerase gamma.

Antigens, Neoplasm↗
PubMed1976

Actions of bleomycin on DNA ligase and polymerases.

Bleomycin inhibited the ligase, which was partially purified from rat ascites hepatoma, AH-130, even at a concentration as low as 0.01-1 mug/ml. The DNA degraded by bleomycin was not repaired by ligase. Therefore, it was suggested that bleomycin at higher concentration produced strand scission of DNA, which could not be repaired by the ligase, and at lower concentration inhibited the ligase reaction presumably by binding to DNA strand or to ligase. Also, the specificity of inhibition by bleomycin on the DNA polymerase of oncogenic RNA virus was tested, comparing with the four kinds of DNA polymerases extracted from the spleen of mice infected with Friend virus. Three kinds of DNA polymerases from spleen were not inhibited by bleomycin, but the fourth enzyme, which was induced in the spleen by virus infection, was inhibited by the antibiotic, when poly-d(AT) and poly-dG with dC were used as template.

Animals↗
PubMed1977

Purification and properties of Rauscher leukemia virus DNA polymerase and selective inhibition of mammalian viral reverse transcriptase by inorganic phosphate.

Rauscher leukemia virus RNA-directed DNA polymerase has been purified to near homogeneity (greater than 90% pure) using affinity chromatography on polycytidylate-agarose with over 85% recovery of input enzymatic activity. The purified enzyme has a molecular weight of approximately 70,000 and appears to consist of a single polypeptide chain. The enzyme is free of DNase, but has RNase H activity. Analysis of the requirements for optimal rates of DNA synthesis by this enzyme using synthetic and natural template-primers has revealed template-specific variations in such requirements. During these studies it was observed that DNA synthesis catalyzed by Rauscher leukemia virus DNA polymerase is inhibited by the addition of inorganic phosphate. An analysis of the mechanism of phosphate inhibition was carried out using the synthetic template-primer poly(A)-(dT)10. It appears that by some mechanism, possibly involving the substrate binding site of the enzyme, phosphate ions inhibit DNA synthesis with a more acute effect on the rate of chain growth than on that of initiation. The extension of these studies to DNA synthesis catalyzed by a variety of mammalian type C viral reverse transcriptases revealed that low levels ( less than or equal to 2 mM) of inorganic phosphate strongly inhibited DNA synthesis. The susceptibility to phosphate inhibition appears unique to mammalian type C viral enzymes since the type B viral enzyme, Escherichia coli DNA polymerase I, avian myeloblastosis virus and Mason Pfizer monkey tumor virus reverse transcriptase and cellular DNA polymerases alpha and gamma are not inhibited by inorganic phosphate. This phenomenon of phosphate inhibition of various DNA polymerases, therefore, provides a new basis for the differentiation of the sources and nature of these enzymes.

Chromatography, Affinity↗