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At least 1,117 records · Page 62Linked to original sources
PubMed1998

Recombination and recombination-dependent DNA replication in bacteriophage T4.

General recombination is essential for growth of phage T4, because origin initiation of DNA replication is inactivated during development, and recombination-dependent initiation is necessary for continuing DNA replication. The requirement of recombination for T4 growth has apparently been a driving force to acquire and maintain multiple recombination mechanisms. This requirement makes this phage an excellent model to analyze several recombination mechanisms that appear redundant under optimal growth conditions but become essential under other conditions, or at different stages of the developmental program. The most important substrate for wild-type T4 recombination is single-stranded DNA generated by incomplete replication of natural or artificial chromosomal ends, or by nucleolytic degradation from induced breaks, or nicks. Recombination circumvents the further erosion of such ends. There are multiple proteins and multiple pathways to initiate formation of recombinants (by single-strand annealing or by strand invasion) and to convert recombinational intermediates into final recombinants ("cut and paste" or "cut and package"), or to initiate extensive DNA replication by "join-copy" or "join-cut-copy" mechanisms. Most T4 recombination is asymmetrical, favoring the initiation of replication. In wild-type T4 these pathways are integrated with physiological changes of other DNA transactions: mainly replication, transcription, and packaging. DNA replication and packaging enzymes participate in recombination, and recombination intermediates supply substrates for replication and packaging. The replicative recombination pathways are also important for transmission of intron DNA to intronless genomes ("homing"), and are implicated in horizontal transfer of foreign genes during evolution of the T-even phages. When horizontal transfer involves heteroduplex formation and repair, it is intrinsically mutagenic and contributes to generation of species barriers between phages.

Bacterial Proteins↗
PubMed1994

The structure of the sleeping genome: implications of sperm DNA organization for somatic cells.

The tertiary structure of the DNA that makes up the eukaryotic genome is remarkably plastic, taking many different forms in response to the different needs of the cell. During the cell cycle of one cell, the DNA is replicated, reorganized into mitotic chromosomes, and decondensed into interphase chromatin. Within one cell at any given point in time, the chromatin is divided into hetero- and euchromatin reflecting active and inactive states of the DNA. This organization varies within one organism since different parts of the genome are active in different cell types. This article focuses on the most dramatic cell-type-specific DNA organization, that found in spermatozoa, in which the entire genome is reorganized into an inactive state that is more highly condensed than mitotic chromosomes. This unique example of eukaryotic DNA organization offers some interesting clues to the still unanswered questions about the role that the three-dimensional packaging of DNA plays in its function.

Animals↗
PubMed1983

Structure and inherent properties of the bacteriophage lambda head shell. IV. Small-head mutants.

Missense mutants of bacteriophage lambda that produce small proheads were found among prophage mutants defective in the major head protein gpE. Measurements of the sedimentation coefficient and molecular weight of the small proheads showed that they have the T = 4 structure composed of 240 molecules of gpE instead of the wild-type T = 7 structure composed of 420 molecules of gpE. When the phage mutants were grown in groE mutants of Escherichia coli, they produced small unprocessed proheads, which contained a smaller number (about 60) of the core protein (gpNu3) molecules than normal unprocessed proheads, which contain about 180 molecules of gpNu3. This shows that the major head protein determines the size of not only the shell but also the core of unprocessed proheads. These mutants by themselves produce very few mature small-headed phage particles, partly because the lambda DNA molecule, whose cos sites are separated at a distance of 48,500 bases, is too long to be packaged into the small proheads. However, the small proheads can package shorter DNA in vivo and in vitro at somewhat reduced efficiency, if the length or a multiple of the length between the cos sites of the DNA is 13,000 to 19,000 bases.

Bacteriophage lambda↗
PubMed2001

Orientations of Tyr 21 and Tyr 24 in the capsid of filamentous virus Ff determined by polarized Raman spectroscopy.

The capsid of filamentous virus Ff is assembled from approximately 2750 copies of a 50-residue alpha-helical subunit, the two tyrosines of which (Tyr 21 and Tyr 24) are located within a hydrophobic sequence that constitutes the subunit interface. We have determined the side chain orientations of Tyr 21 and Tyr 24 by polarized Raman microspectroscopy of oriented Ff fibers, utilizing a novel experimental approach that combines site-specific mutation and residue-specific deuteration of capsid subunits. The polarized Raman signature of Tyr 21 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 21 in an Ff mutant in which Tyr 24 is replaced with methionine. Similarly, the polarized Raman signature of Tyr 24 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 24 in the analogous Tyr 21 --> Met mutant. Polarizations of the corresponding C-D stretching bands in the 2200-2400 cm(-1) interval of the Raman spectrum were measured and interpreted using tensors transferred from a polarized Raman analysis of L-tyrosine-2,3,5,6-d(4) single crystals. Polarized Raman analysis was extended to the bands of Ff near 642 and 855 cm(-1), which originate from vibrational modes of the tyrosine phenolic ring. The results indicate the following: (i) For both Tyr 21 and Tyr 24, the phenolic 2-fold axis (C(1)-C(4) line) is inclined at 41 +/- 5 degrees from the virion axis and the normal to the plane of the phenolic ring is inclined at 71 +/- 5 degrees from the virion axis; (ii) the mutation of Tyr 24, but not the mutation of Tyr 21, perturbs Raman markers of the subunit tryptophan (Trp 26), suggesting interdependence of Tyr 24 and Trp 26 orientations in native Ff; and (iii) polarization anisotropies observed for Raman markers of Ff DNA bases are unperturbed by mutation of either Tyr 21 or Tyr 24, indicating that nonrandom base orientations of packaged Ff DNA are independent of the mutation of either Tyr 21 or Tyr 24. A molecular model consistent with these findings is proposed.

Amino Acid Sequence↗
PubMed1995

Characterization of Serratia entomophila Bacteriophages and the Phage-Resistant Mutant Strain BC4B.

Successful large-scale fermentations of the bacterium Serratia entomophila for use in biological control of the soil-dwelling insect Costelytra zealandica has required the development of a phage-resistant mutant, BC4B. We report our investigations into S. entomophila phages and the nature of the phage resistance mechanism of strain BC4B. The parental strain of BC4B, A1MO2, was found to contain two previously unidentified prophages, (phi)9A and (phi)9B, which were UV inducible and also released spontaneously in large numbers. BC4B was shown to be completely cured of (phi)9A. Single lysogens of (phi)9A and (phi)9B were not homoimmune to any other S. entomophila phages. However, on the basis of DNA-DNA homology, all S. entomophila phages except (phi)CW3 were shown to have significant regions of homology and also packaged their DNA via pac-like mechanisms. The failure of phage particles to adsorb was identified as the basis of phage resistance in BC4B. In addition, it was demonstrated that all known S. entomophila phages are naturally temperature sensitive.

Journal Article↗
PubMed1983

Role of R loops in recA-independent homologous recombination of bacteriophage lambda.

We have previously shown that the DNA-dependent RNA polymerase of Escherichia coli can promote homologous recombination of bacteriophage lambda independently of the recA function. To detect this recombination, we jointly infected the cells with a pair of lambda phages in the presence of chloramphenicol, extracted intracellular lambda DNA molecules, packaged them in vitro, and measured the number of resulting recombinant phage particles. We showed that the recombination of DNA molecules takes place in vitro after extraction of DNA from the cells. We fractionated recombinogenic forms of intracellular lambda DNA and showed that they carry RNA. These and other results suggest that the R (RNA) loop structures, generated by transcription within the cells, promote homologous recombination in vitro. We discuss possible mechanisms of this recombination and compare those with the other forms of general recombination.

Bacteriophage lambda↗
PubMed2001

Reconstitution of chromatin in vitro.

It is now generally believed that DNA methylation is responsible for genomic imprinting in mammals (1). Recent experimental evidence has provided an elegant mechanism for repression of gene expression by DNA methylation. This evidence suggests that proteins that recognize specifically methylated CpGs may contribute to the formation of inactive chromatin (2-5). Nucleosomes are the basic unit of chromatin, consisting of a core of 146 bp of DNA wrapped around a histone octamer (two molecules of each of H2A, H2B, H3, and H4) and a stretch of linker DNA between adjacent nucleosomes. The binding of a fifth histone, known as a linker histone or H1, promotes the packaging of strings of nucleosomes into a 30 nm chromatin fiber (6). Packaging of DNA into nucleosomes and the chromatin fiber greatly restricts the availability of the DNA for nuclear processes such as transcription.

Chromatin↗
PubMed1999

Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd.

Pf1, a class II filamentous virus, has been investigated by ultraviolet resonance Raman (UVRR) spectroscopy with excitation wavelengths of 257, 244, 238, and 229 nm. The 257-nm UVRR spectrum is rich in Raman bands of the packaged single-stranded DNA (ssDNA) genome, despite the low DNA mass (6%) of the virion. Conversely, the 229-nm UVRR spectrum is dominated by tyrosines (Tyr 25 and Tyr 40) of the 46-residue alpha-helical coat subunit. UVRR spectra excited at 244 and 238 nm exhibit Raman bands diagnostic of both viral DNA and coat protein tyrosines. Raman markers of packaged Pf1 DNA contrast sharply with those of the DNA packaged in the class I filamentous virus fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J., Jr. (1997) Biochemistry 36, 7810-7820]. Interestingly, deoxynucleotides of Pf1 DNA exhibit sugars in the C2'-endo/anti conformation and bases that are largely unstacked, compared with C3'-endo/anti conformers and very strong base stacking in fd DNA; hydrogen-bonding interactions of thymine carbonyls are also different in Pf1 and fd. On the other hand, coat protein tyrosines of Pf1 exhibit Raman markers of ring environment identical to those of fd, including an anomalous singlet at 853 cm-1 in lieu of the canonical Fermi doublet (850/830 cm-1) found in globular proteins. The results indicate markedly different modes of organization of ssDNA in Pf1 and fd virions, despite similar environments for coat protein tyrosines, and suggest strong hydrogen-bonding interactions between DNA bases and coat subunits of Pf1 but not between those of fd. We propose that structural relationships between the protein coat and encapsidated ssDNA genome are also fundamentally different in the two assemblies.

Capsid↗
PubMed1992

DNA Translator and Aligner: HyperCard utilities to aid phylogenetic analysis of molecules.

DNA Translator and Aligner are molecular phylogenetics HyperCard stacks for Macintosh computers. They manipulate sequence data to provide graphical gene mapping, conversions, translations and manual multiple-sequence alignment editing. DNA Translator is able to convert documented GenBank or EMBL documented sequences into linearized, rescalable gene maps whose gene sequences are extractable by clicking on the corresponding map button or by selection from a scrolling list. Provided gene maps, complete with extractable sequences, consist of nine metazoan, one yeast, and one ciliate mitochondrial DNAs and three green plant chloroplast DNAs. Single or multiple sequences can be manipulated to aid in phylogenetic analysis. Sequences can be translated between nucleic acids and proteins in either direction with flexible support of alternate genetic codes and ambiguous nucleotide symbols. Multiple aligned sequence output from diverse sources can be converted to Nexus, Hennig86 or PHYLIP format for subsequent phylogenetic analysis. Input or output alignments can be examined with Aligner, a convenient accessory stack included in the DNA Translator package. Aligner is an editor for the manual alignment of up to 100 sequences that toggles between display of matched characters and normal unmatched sequences. DNA Translator also generates graphic displays of amino acid coding and codon usage frequency relative to all other, or only synonymous, codons for approximately 70 select organism-organelle combinations. Codon usage data is compatible with spreadsheet or UWGCG formats for incorporation of additional molecules of interest. The complete package is available via anonymous ftp and is free for non-commercial uses.

Amino Acid Sequence↗
PubMed1968

Deoxyribonucleic acid synthesis in FV-3-infected mammalian cells.

Deoxyribonucleic acid (DNA) synthesis and virus growth in frog virus 3 (FV-3)-infected mammalian cells in suspension were examined. The kinetics of thymidine incorporation into DNA was followed by fractionating infected cells. The cell fractionation procedure separated replicating viral DNA from matured virus. Incorporation of isotope into the nuclear fraction was depressed 2 to 3 hr postinfection; this inhibition did not require protein synthesis. About 3 to 4 hr postinfection, there was an increase in thymidine incorporation into both nuclear and cytoplasmic fractions. The nuclear-associating DNA had a guanine plus cytosine (GC) content of 52%; unlike host DNA it was synthesized in the presence of mitomycin C, it could be removed from nuclei by centrifugation through sucrose, and it was susceptible to nuclease digestion. This nuclear-associating DNA appeared to be a precursor of cytoplasmic DNA of infected cells. The formation of the latter DNA class could be selectively inhibited by conditions (infection at 37 C or inhibition of protein synthesis) that permit continued incorporation of thymidine into nuclear-associating DNA. The cytoplasmic DNA class also had a GC content of 52%, was resistant to nuclease degradation, and its sedimentation profile in sucrose gradients corresponded to that of infective virus. Contrary to previous reports, we found that (i) viral DNA synthesis can continue in the absence of concomitant protein synthesis, and (ii) viral DNA synthesis is not abolished at 37 C. The temperature lesion in FV-3 replication appeared to be in the packaging of DNA into the form that appears in the cytoplasmic fraction of disrupted cells.

Animals↗
PubMed1980

T1 genes which affect transduction.

Amber mutants of T1 were grown on each of three donor strains which were identical except that they carried different suppressors: respectively, supD, supE, and supB. The efficiency with which the mutants were able to transduce was tested after growth on each donor. In general, it was found that functions which control the synthesis of phage DNA usually caused significant increases in the efficiency of transduction (EOT). A few mutants located in genes essential for head production caused significant decreases in EOT. The presence of a particular suppressor in a donor can cause noteworthy changes in the EOT by certain of the mutant phages. Amber mutations in gene 3 of T1 were extremely sensitive to the particular suppressor present in the donor, showing a 17-fold decrease in EOT compared with other mutants after growth in donors with the supD suppressor and a 75-fold increase after growth in supE donors. Increases in EOT by early genes of T1 do not seem to be caused by a lack of competition of bacterial DNA with phage DNA during packaging since, in most instances, infective phage were produced in relatively normal amounts compared with wild-type T1. Phage DNA synthesis and degradations of the host chromosome are closely coupled in T1 infections; we believe that increases in EOT by mutants of early functions are due to inefficient degradation of the host chromosome.

DNA, Viral↗
PubMed2005

An epigenetic code for DNA damage repair pathways?

Exposure of living cells to intracellular or external mutagens results in DNA damage. Accumulation of DNA damage can lead to serious consequences because of the deleterious mutation rate resulting in genomic instability, cellular senescence, and cell death. To counteract genotoxic stress, cells have developed several strategies to detect defects in DNA structure. The eukaryotic genomic DNA is packaged through histone and nonhistone proteins into a highly condensed structure termed chromatin. Therefore the cellular enzymatic machineries responsible for DNA replication, recombination, and repair must circumvent this natural barrier in order to gain access to the DNA. Several studies have demonstrated that histone/chromatin modifications such as acetylation, methylation, and phosphorylation play crucial roles in DNA repair processes. This review will summarize the recent data that suggest a regulatory role of the epigenetic code in DNA repair processes. We will mainly focus on different covalent reversible modifications of histones as an initial step in early response to DNA damage and subsequent DNA repair. Special focus on a potential epigenetic histone code for these processes will be given in the last section. We also discuss new technologies and strategies to elucidate the putative epigenetic code for each of the DNA repair processes discussed.

Adenosine Diphosphate Ribose↗
PubMed2006

Topical DermaVir vaccine targeting dendritic cells.

DermaVir employs a topical, non-invasive method for vaccine delivery to dendritic cells. The vaccine product contains plasmid DNA as the active ingredient, encoding authentically expressed retroviral genes with appropriate safety modifications. The non-viral delivery system packages the DNA within pathogen-like nanoparticles and studies indicate that vaccine antigens are taken up by epidermal Langerhans cells, the precursors of dendritic cells. DermaVir loaded dendritic cells reach the draining lymph node target but not the bloodstream nor indiscriminately other organ systems. Safety data from DermaVir immunized infected macaques indicate improved survival, absence of apparent toxicities other than transient erythema and lack of recombination between the vaccine DNA and the infectious viral DNA integrated in the host genome. DermaVir represents a potential new approach for the treatment of HIV infection to be utilized either in conjunction with antiretroviral therapy or during structured treatment interruption.

AIDS Vaccines↗
PubMed2007

Formation of a multiple protein complex on the adenovirus packaging sequence by the IVa2 protein.

During adenovirus virion assembly, the packaging sequence mediates the encapsidation of the viral genome. This sequence is composed of seven functional units, termed A repeats. Recent evidence suggests that the adenovirus IVa2 protein binds the packaging sequence and is involved in packaging of the genome. Study of the IVa2-packaging sequence interaction has been hindered by difficulty in purifying the protein produced in virus-infected cells or by recombinant techniques. We report the first purification of a recombinant untagged version of the adenovirus IVa2 protein and characterize its binding to the packaging sequence in vitro. Our data indicate that there is more than one IVa2 binding site within the packaging sequence and that IVa2 binding to DNA requires the A-repeat consensus, 5'-TTTG-(N(8))-CG-3'. Furthermore, we present evidence that IVa2 forms a multimeric complex on the packaging sequence. These data support a model in which adenovirus DNA packaging occurs via the formation of a IVa2 multiprotein complex on the packaging sequence.

Adenoviridae↗
PubMed2006

DNA translocation and loop formation mechanism of chromatin remodeling by SWI/SNF and RSC.

ATP-dependent chromatin-remodeling complexes (remodelers) modulate gene transcription by regulating the accessibility of highly packaged genomic DNA. However, the molecular mechanisms involved at the nucleosomal level in this process remain controversial. Here, we monitor the real-time activity of single ySWI/SNF or RSC complexes on single, stretched nucleosomal templates under tensions above 1 pN forces. We find that these remodelers can translocate along DNA at rates of approximately 13 bp/s and generate forces up to approximately 12 pN, producing DNA loops of a broad range of sizes (20-1200 bp, average approximately 100 bp) in a nucleosome-dependent manner. This nucleosome-specific activity differs significantly from that on bare DNA observed under low tensions and suggests a nucleosome-remodeling mechanism through intranucleosomal DNA loop formation. Such loop formation may provide a molecular basis for the biological functions of remodelers.

Adenosine Triphosphate↗
PubMed1990

DNA methylation. The effect of minor bases on DNA-protein interactions.

DNA methylation is found almost ubiquitously in nature and the methyltransferases show evidence of a common evolutionary origin. It will be a fascinating study in protein evolution to follow the ways in which the structures of the various enzymes have developed. Although methylation may have a direct effect on DNA structure the evidence for the importance of this in vivo is accumulating only slowly. In contrast, there is now abundant evidence that methylation of DNA affects DNA-protein interactions and so may have a function in all processes in which such interactions occur. The binding of nucleases is affected in the processes of mismatch repair, DNA restriction and possibly demethylation during differentiation in vertebrates. The binding of transcription factors is affected by DNA methylation and the association of DNA with packaging and segregation proteins may play a part in the control of transcription and replication. The interplay of these effects makes DNA methylation a complex but rewarding area for study. Perhaps we should no longer refer to methylcytosine and methyladenine as minor bases, but rather as key bases which help regulate the functions of DNA.

DNA↗
PubMed1990

Herpes simplex virus type 1 UL28 gene product is important for the formation of mature capsids.

The herpes simplex virus type 1 temperature-sensitive (ts) DNA-positive mutant ts1203 has been characterized. The ts lesion in ts1203 was located by marker rescue within the coding region of gene UL28. Nuclei of cells infected with ts1203 at the non-permissive temperature (NPT) contained large numbers of capsids with a uniform morphology. These capsids lacked DNA but had a defined internal structure. No full capsids were detected at the NPT, suggesting that ts1203 was unable to package viral DNA. In this respect ts1203 is similar to ts1201 which has a defect in gene UL26. The capsids made by ts1203 at the NPT, however, contained a more compact internal structure than those of ts1201. In addition, ts1203 capsids were dispersed throughout the nucleus whereas ts1201 capsids were frequently found clustered together in large arrays. Southern blot and sedimentation analyses of viral DNA confirmed that ts1203 had an encapsidation defect and showed that most of the mutant DNA at the NPT was of a high Mr. The effect of the ts1203 mutation could not be reversed in the absence of de novo protein synthesis by transferring mutant-infected cells from the NPT to the permissive temperature.

Blotting, Southern↗
PubMed1995

In vitro assembly of infectious virions of double-stranded DNA phage phi 29 from cloned gene products and synthetic nucleic acids.

Up to 6 x 10(7) PFU of infectious virions of the double-stranded DNA bacteriophage phi 29 per ml were assembled in vitro, with 11 proteins derived from cloned genes and nucleic acids synthesized separately. The genomic DNA-gp3 protein conjugate was efficiently packaged into a purified recombinant procapsid with the aid of a small viral RNA (pRNA) transcript, a DNA-packaging ATPase (gp16), and ATP. The DNA-filled capsids were subsequently converted into infectious virions after the addition of four more recombinant proteins for neck and tail assembly. Electron microscopy and genome restriction mapping confirmed the identity of the infectious phi 29 virions synthesized in this system. A nonstructural protein, gp13, was indispensable for the assembly of infectious virions. The overproduced tail protein gp9 was present in solution in mostly dimeric form and was purified to homogeneity. The purified gp9 was biologically active for in vitro phi 29 assembly. Higher-order concentration dependence of in vitro phi 29 assembly on gp9 suggests that a complete tail did not form before attaching to the DNA-filled capsid, a result contrary to earlier findings for phages T4 and lambda. The work described here constitutes an extremely sensitive assay system for the analysis of components in phi 29 assembly and dissection of functional domains of structural components, enzymes, and pRNA (C.-S. Lee and P. Guo, Virology 202:1039-1042, 1995). Efficient packaging of foreign DNA in vitro and synthesis of viral particles from recombinant proteins facilitate the development of phi 29 as an in vivo gene delivery system. The finding that purified tail protein was able to incorporate into infectious virions might allow the construction of chimeric phi 29 carrying a tail fused to ligands for specific receptor of human cells.

Bacillus Phages↗