Secondary literature sources for Bro-N
The following references were automatically generated.
- Boyle KA, Greseth MD, Traktman P
- Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication.
- J Virol. 2015; 89: 6312-27
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The duplication of the poxvirus double-stranded DNA genome occurs in cytoplasmic membrane-delimited factories. This physical autonomy from the host nucleus suggests that poxvirus genomes encode the full repertoire of proteins committed for genome replication. Biochemical and genetic analyses have confirmed that six viral proteins are required for efficient DNA synthesis; indirect evidence has suggested that the multifunctional H5 protein may also have a role. Here we show that H5 localizes to replication factories, as visualized by immunofluorescence and immunoelectron microscopy, and can be retrieved upon purification of the viral polymerase holoenzyme complex. The temperature-sensitive (ts) mutant Dts57, which was generated by chemical mutagenesis and has a lesion in H5, exhibits defects in DNA replication and morphogenesis under nonpermissive conditions, depending upon the experimental protocol. The H5 variant encoded by the genome of this mutant is ts for function but not stability. For a more precise investigation of how H5 contributes to DNA synthesis, we placed the ts57 H5 allele in an otherwise wild-type viral background and also performed small interfering RNA-mediated depletion of H5. Finally, we generated a complementing cell line, CV-1-H5, which allowed us to generate a viral recombinant in which the H5 open reading frame was deleted and replaced with mCherry (vDeltaH5). Analysis of vDeltaH5 allowed us to demonstrate conclusively that viral DNA replication is abrogated in the absence of H5. The loss of H5 does not compromise the accumulation of other early viral replication proteins or the uncoating of the virion core, suggesting that H5 plays a direct and essential role in facilitating DNA synthesis. IMPORTANCE: Variola virus, the causative agent of smallpox, is the most notorious member of the Poxviridae family. Poxviruses are unique among DNA viruses that infect mammalian cells, in that their replication is restricted to the cytoplasm of the cell. This physical autonomy from the nucleus has both cell biological and genetic ramifications. Poxviruses must establish cytoplasmic niches that support replication, and the genomes must encode the repertoire of proteins necessary for genome synthesis. Here we focus on H5, a multifunctional and abundant viral protein. We confirm that H5 associates with the DNA polymerase holoenzyme and localizes to the sites of DNA synthesis. By generating an H5-expressing cell line, we were able to isolate a deletion virus that lacks the H5 gene and show definitively that genome synthesis does not occur in the absence of H5. These data support the hypothesis that H5 is a crucial participant in cytoplasmic poxvirus genome replication.
- Krajewski WA, Nakamura T, Mazo A, Canaani E
- A motif within SET-domain proteins binds single-stranded nucleic acids and transcribed and supercoiled DNAs and can interfere with assembly of nucleosomes.
- Mol Cell Biol. 2005; 25: 1891-9
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The evolutionary conserved SET domain is present in many eukaryotic chromatin-associated proteins, including some members of the trithorax (TrxG) group and the polycomb (PcG) group of epigenetic transcriptional regulators and modifiers of position effect variegation. All SET domains examined exhibited histone lysine methyltransferase activity, implicating these proteins in the generation of epigenetic marks. However, the mode of the initial recruitment of SET proteins to target genes and the way that their association with the genes is maintained after replication are not known. We found that SET-containing proteins of the SET1 and SET2 families contain motifs in the pre-SET region or at the pre-SET-SET and SET-post-SET boundaries which very tightly bind single-stranded DNA (ssDNA) and RNA. These motifs also bind stretches of ssDNA generated by superhelical tension or during the in vitro transcription of duplex DNA. Importantly, such binding withstands nucleosome assembly, interfering with the formation of regular nucleosomal arrays. Two representatives of the SUV39 SET family, SU(VAR)3-9 and G9a, did not bind ssDNA. The trxZ11 homeotic point mutation, which is located within TRX SET and disrupts embryonic development, impairs the ssDNA binding capacity of the protein. We suggest that the motifs described here may be directly involved in the biological function(s) of SET-containing proteins. The binding of single-stranded nucleic acids might play a role in the initial recruitment of the proteins to target genes, in the maintenance of their association after DNA replication, or in sustaining DNA stretches in a single-stranded configuration to allow for continuous transcription.
- Song WJ, Qin QW, Qiu J, Huang CH, Wang F, Hew CL
- Functional genomics analysis of Singapore grouper iridovirus: complete sequence determination and proteomic analysis.
- J Virol. 2004; 78: 12576-90
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Here we report the complete genome sequence of Singapore grouper iridovirus (SGIV). Sequencing of the random shotgun and restriction endonuclease genomic libraries showed that the entire SGIV genome consists of 140,131 nucleotide bp. One hundred sixty-two open reading frames (ORFs) from the sense and antisense DNA strands, coding for lengths varying from 41 to 1,268 amino acids, were identified. Computer-assisted analyses of the deduced amino acid sequences revealed that 77 of the ORFs exhibited homologies to known virus genes, 23 of which matched functional iridovirus proteins. Forty-two putative conserved domains or signatures were detected in the National Center for Biotechnology Information CD-Search database and PROSITE database. An assortment of enzyme activities involved in DNA replication, transcription, nucleotide metabolism, cell signaling, etc., were identified. Viruses were cultured on a cell line derived from the embryonated egg of the grouper Epinephelus tauvina, isolated, and purified by sucrose gradient ultracentrifugation. The protein extract from the purified virions was analyzed by polyacrylamide gel electrophoresis followed by in-gel digestion of protein bands. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and database searching led to identification of 26 proteins. Twenty of these represented novel or previously unidentified genes, which were further confirmed by reverse transcription-PCR (RT-PCR) and DNA sequencing of their respective RT-PCR products.
- Villarreal LP, DeFilippis VR
- A hypothesis for DNA viruses as the origin of eukaryotic replication proteins.
- J Virol. 2000; 74: 7079-84
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The eukaryotic replicative DNA polymerases are similar to those of large DNA viruses of eukaryotic and bacterial T4 phages but not to those of eubacteria. We develop and examine the hypothesis that DNA virus replication proteins gave rise to those of eukaryotes during evolution. We chose the DNA polymerase from phycodnavirus (which infects microalgae) as the basis of this analysis, as it represents a virus of a primitive eukaryote. We show that it has significant similarity with replicative DNA polymerases of eukaryotes and certain of their large DNA viruses. Sequence alignment confirms this similarity and establishes the presence of highly conserved domains in the polymerase amino terminus. Subsequent reconstruction of a phylogenetic tree indicates that these algal viral DNA polymerases are near the root of the clade containing all eukaryotic DNA polymerase delta members but that this clade does not contain the polymerases of other DNA viruses. We consider arguments for the polarity of this relationship and present the hypothesis that the replication genes of DNA viruses gave rise to those of eukaryotes and not the reverse direction.
- Bardoni B, Schenck A, Mandel JL
- A novel RNA-binding nuclear protein that interacts with the fragile X mental retardation (FMR1) protein.
- Hum Mol Genet. 1999; 8: 2557-66
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Silenced expression of the FMR1 gene is responsible for the fragile X syndrome. The FMR1 gene codes for an RNA binding protein (FMRP), which can shuttle between the nucleus and the cytoplasm and is found associated to polysomes in the cytoplasm. By two-hybrid assay in yeast, we identified a novel protein interacting with FMRP: nuclear FMRP interacting protein (NUFIP). NUFIP mRNA expression is strikingly similar to that of the FMR1 gene in neurones of cortex, hippocampus and cerebellum. At the subcellular level, NUFIP colocalizes with nuclear isoforms of FMRP in a dot-like pattern. NUFIP presents a C2H2 zinc finger motif and a nuclear localization signal, but has no homology to known proteins and shows RNA binding activity in vitro. NUFIP does not interact with the FMRP homologues encoded by the FXR1 and FXR2 genes. Thus, these results indicate a specific nuclear role for FMRP.
- Altschul SF et al.
- Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
- Nucleic Acids Res. 1997; 25: 3389-402
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The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position-Specific Iterated BLAST (PSI-BLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST is used to uncover several new and interesting members of the BRCT superfamily.
- Seeberg E, Eide L, Bjoras M
- The base excision repair pathway.
- Trends Biochem Sci. 1995; 20: 391-7
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The base excision repair pathway has evolved to protect cells from the deleterious effects of endogenous DNA damage induced by hydrolysis, reactive oxygen species and other intracellular metabolites that modify DNA base structure. However, base excision repair is also important to resist lesions produced by ionizing radiation and strong alkylating agents, which are similar to those induced by endogenous factors.
- Millns AK, Carpenter MS, DeLange AM
- The vaccinia virus-encoded uracil DNA glycosylase has an essential role in viral DNA replication.
- Virology. 1994; 198: 504-13
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The vaccinia virus conditional-lethal temperature-sensitive (ts) mutant ts4149 is, at the nonpermissive temperature, severely impaired in its ability to replicate its DNA genome. Compared to wild type, the amount of replication is suppressed by several orders of magnitude, and the little DNA that is replicated is not converted to mature linear genomes. We have demonstrated that this "DNA-" phenotype is not the result of a failure to produce early proteins. In agreement with the DNA- phenotype, intermediate and late gene expression were not detected. Marker rescue and DNA sequencing located the mutation in ts4149 to open reading frame D4. This gene has recently been shown to encode a 25-kDa protein with uracil DNA glycosylase activity (D. T. Stuart, C. Upton, M. A. Higman, E. G. Niles, and G. McFadden (1993), J. Virol. 67, 2503-2512). We speculate on the function of this "essential" viral repair enzyme and its role(s) in viral DNA replication.
- Ravanello MP, Hruby DE
- Conditional lethal expression of the vaccinia virus L1R myristylated protein reveals a role in virion assembly.
- J Virol. 1994; 68: 6401-10
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Within vaccinia virus-infected cells, the product of the L1R open reading frame is covalently modified by myristic acid at the penultimate NH2-terminal glycine residue. Previously we have shown that while the L1R protein is a constituent of both intracellular mature virus particles and extracellular enveloped virions which are released from the infected cell, it is associated exclusively with the primary membranes surrounding the virion core. Given this rather specific localization, it was of interest to study the potential role of this essential gene in virus replication and morphogenesis. To this end, we have constructed a recombinant vaccinia virus in which expression of the L1R gene can be transcriptionally repressed. Without the inducer isopropylthiogalactopyranoside (IPTG), synthesis of the L1R protein was blocked, resulting in a total inhibition of plaque formation. Velocity sedimentation of viral particles labeled in the presence of [3H]thymidine, grown in the absence of IPTG, revealed a substantial reduction in viral DNA incorporation into virions. Likewise, proteolysis of the major core proteins p4a, p4b, and p25K, believed to occur during the final stages of virion maturation, was severely impaired. In the absence of L1R expression, only immature virions could be detected by electron microscopy. Transient expression of a plasmid containing the full-length L1R gene driven by its own promoter was able to complement and rescue the defective phenotype. However, a plasmid bearing a mutation in the myristyl acceptor glycine residue was unable to biologically rescue the recombinant, and the protein was not detected in purified virions.trans complementation using a truncated, myristylated form of the L1R protein partially rescued the defective mutant. Collectively, these data suggest that myristic acid mediates essential interactions of the L1R protein with viral membranes and/or other virion components that lead to the productive assembly, maturation, and release of particles.
- Stuart DT, Upton C, Higman MA, Niles EG, McFadden G
- A poxvirus-encoded uracil DNA glycosylase is essential for virus viability.
- J Virol. 1993; 67: 2503-12
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Infection of cultured mammalian cells with the Leporipoxvirus Shope fibroma virus (SFV) causes the induction of a novel uracil DNA glycosylase activity in the cytoplasms of the infected cells. The induction of this activity, early in infection, correlates with the early expression of the SFV BamHI D6R open reading frame which possesses significant protein sequence similarity to eukaryotic and prokaryotic uracil DNA glycosylases. The SFV BamHI D6R open reading frame and the homologous HindIII D4R open reading frame from the Orthopoxvirus vaccinia virus were cloned under the regulation of a phage T7 promoter and expressed in Escherichia coli as insoluble high-molecular-weight aggregates. During electrophoresis on sodium dodecyl sulfate-polyacrylamide gels, the E. coli-expressed proteins migrate with an apparent molecular mass of 25 kDa. The insoluble protein aggregate generated by expression in E. coli was solubilized in urea and, following a subsequent refolding step, displayed the ability to excise uracil residues from double-stranded plasmid DNA substrates, with the subsequent formation of apyrimidinic sites. The viral enzyme, like all other characterized uracil DNA glycosylases, is active in the presence of high concentrations of EDTA, is substrate inhibited by uracil, and does not display any endonuclease activity. Attempts to inactivate the HindIII D4R gene of vaccinia virus by targeted insertion of a dominant xanthine-guanine phosphoribosyltransferase selection marker or direct insertion of a frame-shifted oligonucleotide were uniformly unsuccessful demonstrating that, unlike the uracil DNA glycosylase described for herpesviruses, the poxvirus enzyme is essential for virus viability.