African swine fever virus (ASFV) encodes a novel DNA polymerase, constituted of only 174 amino acids, belonging to the polymerase (pol) X family of DNA polymerases. Biochemical analyses of the purified enzyme indicate that ASFV pol X is a monomeric DNA-directed DNA polymerase, highly distributive, lacking a proofreading 3-5-exonuclease, and with a poor discrimination against dideoxynucleotides. A multiple alignment of family X DNA polymerases, together with the extrapolation to the crystal structure of mammalian DNA polymerase  (pol ), showed the conservation in ASFV pol X of the most critical residues involved in DNA binding, nucleotide binding, and catalysis of the polymerization reaction. Therefore, the 20-kDa ASFV pol X most likely represents the minimal functional version of an evolutionarily conserved pol -type DNA polymerase core, constituted by only the "palm" and "thumb" subdomains. It is worth noting that such an "unfingered" DNA polymerase is able to handle templated DNA polymerization with a considerable high fidelity at the base discrimination level. Base excision repair is considered to be a cellular defense mechanism repairing modified bases in DNA. Interestingly, the fact that ASFV pol X is able to conduct filling of a single nucleotide gap points to a putative role in base excision repair during the ASFV life cycle.Despite the variety of existing DNA polymerases, there are a few basic principles that are common to all these enzymes, irrespective of their role either in DNA replication or in DNA repair. The basic chemistry of each individual reaction always involves a pair of divalent metal ions that are coordinated by carboxylate residues. Such a two-metal ion mechanism, originally proposed by Beese and Steitz (1) and probably extrapolative to all nucleotidyltransferases (2-4), appears to be either evolutionarily conserved or acquired by convergent evolution of nonhomologous proteins. In addition to the general deoxynucleotidyl transfer mechanism, it appears that some structural convergency could apply also for the interaction with DNA, a common substrate; an overall view of the crystal structures available for DNA-dependent polymerases always shows a hand-shaped structure, with "thumb," "palm," and "fingers" subdomains, defining at least one cleft for holding DNA (5-12). Moreover, both DNA replicases and DNA repair enzymes are often multienzymatic proteins, having built-in nucleolytic activities that exist as individual structural modules, separated from the polymerization domain. Thus, the paradigmatic Escherichia coli DNA polymerase I (pol I) 1 has a proofreading 3Ј-5Ј-exonuclease, and a 5Ј-3Ј-exonuclease to remove the RNA from the Okazaki fragments (13). Most DNA replicases are also endowed with a proofreading 3Ј-5Ј-exonuclease domain, having an evolutionarily conserved pol I-type active site (14). Reverse transcriptases have an RNase H activity, required for second strand DNA synthesis (reviewed in Telesnitsky and Goff (15)). Even pol , the smallest of the known DNA polymerases (39 kDa)...
The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg2+, the preferred cation, and has an apparent Km for dUTP of 1 μM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vΔE165R) has been obtained. Growth kinetics showed that vΔE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.
We report the identification and functional characterization of ariadne-1 (ari-1), a novel and vital Drosophila gene required for the correct differentiation of most cell types in the adult organism. Also, we identify a sequence-related gene, ari-2, and the corresponding mouse and human homologues of both genes. All these sequences define a new protein family by the Acid-rich, RING finger, B-box, RING finger, coiled-coil (ARBRCC) motif string. In Drosophila, ari-1 is expressed throughout development in all tissues. The mutant phenotypes are most noticeable in cells that undergo a large and rapid membrane deposition, such as rewiring neurons during metamorphosis, large tubular muscles during adult myogenesis, and photoreceptors. Occasional survivors of null alleles exhibit reduced life span, motor impairments, and short and thin bristles. Single substitutions at key cysteines in each RING finger cause lethality with no survivors and a drastic reduction of rough endoplasmic reticulum that can be observed in the photoreceptors of mosaic eyes. In yeast two-hybrid assays, the protein ARI-1 interacts with a novel ubiquitin-conjugating enzyme, UbcD10, whose sequence is also reported here. The N-terminal RING-finger motif is necessary and sufficient to mediate this interaction. Mouse and fly homologues of both ARI proteins and the Ubc can substitute for each other in the yeast two-hybrid assay, indicating that ARI represents a conserved novel mechanism in development. In addition to ARI homologues, the RBR signature is also found in the Parkinson-disease-related protein Parkin adjacent to an ubiquitin-like domain, suggesting that the study of this mechanism could be relevant for human pathology.
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