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PubMed2004

Chromatin modifiers in transcription and DNA repair.

In eukaryotes, the packaging of DNA into nucleosomes and the organization of chromatin fibres generate constraints for all nuclear processes involving DNA, including replication, repair, recombination and transcription. The three major processes that regulate chromatin structure and counterbalance its repressive effects are ATP-dependent chromatin remodeling, post-translational modification of histones and histone replacement. While many of these processes have been intensively studied with respect to their effects on transcription, there is also evidence that they affect other nuclear processes involving DNA. This review explores the functions of chromatin-remodeling factors and histone-modifying enzymes in gene regulation and summarizes recent findings which suggest a role for these chromatin modifiers in DNA repair.

Animals↗

PubMed1998

DNA requirements in vivo for phage T4 packaging.

Phage T4 terminase, comprising the products of genes 16 and 17, packages headfuls of DNA from a concatemer but its mechanism of DNA recognition remains to be determined. Phage T4 terminase gene sequences were introduced into prophage lambda imm434 and plasmids in order to assess their effect on packaging as measured by transduction frequency and DNA content of T4-transducing particles. Multiple copy prophage lambda imm434 genes were transduced at 100-fold higher frequency, and high copy plasmids were transduced at 1000-fold higher frequency than single copy prophage or chromosomal genes T4 16 gene inserts enhanced both prophage and plasmid packaging; terminase gene-containing plasmid DNA in T4 transducing particles could exceed 10% of the total. Deletion or base change of the 24-bp gene 16 3' region which is required for sequence specific amplification of terminase gene 17 (Hp 17 mutations) depressed these elevated plasmid transduction frequencies, suggesting that this is a preferred T4 pac sequence. Moreover, a specific gene 16-containing pac fragment could be detected in mature, packaged phage T4 DNA following restriction endonuclease digestion. We conclude that both the copy number of homologous sequences and the DNA pac sequence(s) themselves are important for packaging, consistent with a synapsis model for regulation of terminase cutting and packaging in phage T4.

Bacteriophage T4↗

PubMed2003

The product of the bacteriophage lambda W gene: purification and properties.

Gene W is one of the 10 genes that control the morphogenesis of the bacteriophage lambda head. The morpho genesis of the phage lambda head proceeds through the synthesis of an intermediate assembly called the prohead. This is an empty shell into which the bacteriophage DNA is introduced--packaged--by the phage enzyme DNA terminase. The product of W (gpW) acts after DNA packaging, but before the addition of another phage product, gene product FII, and before the addition of tails. The role of gpW is unknown. The structure of N- and C-tagged gpW has been previously determined by nuclear magnetic resonance (NMR) spectroscopy. Here we report some of the properties of the native protein. The purification of gpW to homogeneity, overproduced by a plasmid derivative, is described. To obtain large amounts of the protein, the ribosome-binding site had to be modified, showing that inefficient translation of the message is the main mechanism limiting W gene expression. The molecular weight of the protein is in close agreement to the value predicted from the DNA sequence of the gene, which suggests that it is not post-transcriptionally modified. It behaves as a monomer in solution. Radioactively labeled gpW is incorporated into phage particles in in vitro complementation, showing that gpW is a structural protein. The stage at which gpW functions and other circumstantial evidence support the idea that six molecules of gpW polymerize on the connector before the incorporation of six molecules of gpFII and before the tail attaches.

Bacteriophage lambda↗

PubMed2003

Structure of a viral DNA gatekeeper at 10 A resolution by cryo-electron microscopy.

In tailed bacteriophages and herpes viruses, the viral DNA is packaged through the portal protein channel. Channel closure is essential to prevent DNA release after packaging. Here we present the connector structure from bacteriophage SPP1 using cryo-electron microscopy and single particle analysis. The multiprotein complex comprises the portal protein gp6 and the head completion proteins gp15 and gp16. Although we show that gp6 in the connector has a fold similar to that of the isolated portal protein, we observe conformational changes in the region of gp6 exposed to the DNA-packaging ATPase and to gp15. This reorganization does not cause closure of the channel. The connector channel traverses the full height of gp6 and gp15, but it is closed by gp16 at the bottom of the complex. Gp16 acts as a valve whose closure prevents DNA leakage, while its opening is required for DNA release upon interaction of the virus with its host.

Adenosine Triphosphatases↗

PubMed1995

DNA Strider. An inexpensive sequence analysis package for the Macintosh.

DNA Strider is a user-friendly Macintosh program designed for analysis of molecular sequence data. The program shows great versatility in choice of genetic codes, restriction enzymes, algorithms for hydropathy plots, and interconversions between types of sequence data. It is very straightforward and interacts well with other sequence and phylogenetic analysis packages, importing and exporting data easily. The graphic outputs can be used directly for manuscript-quality figures, or exported to more sophisticated text or graphic programs.

Amino Acid Sequence↗

PubMed1984

The maturation of coliphage lambda DNA in the absence of its packaging.

In vivo, lambda DNA cannot be cleaved at cos (matured) if proheads are not present; in vitro, however, cos cleavage readily takes place in the absence of proheads. In order to investigate this paradox, we have constructed plasmids that synthesize lambda terminase in vivo upon induction. The plasmids also contain cos at the normal position, about 190 bp upstream of lambda gene Nul. One of the plasmids, pFM3, produces levels of terminase comparable to those found after phage induction. If cells carrying pFM3 are thermoinduced, almost 100% of the intracellular plasmid DNA has a double-strand interruption at or near cos. Since the only lambda genes that pFM3 carries are Nul, A, W and B, this in vivo cleavage is occurring in the absence of proheads. Previous failure to observe lambda maturation with phages carrying prohead mutations may be due to exonucleolytic degradation of the unprotected DNA ends, a different DNA topology or compartmentalization, or terminase inhibition in the absence of prohead by the product of another lambda gene that maps to the right of gene B.

Bacteriophage lambda↗

PubMed2004

Peculiarities of the DNA of MM1, a temperate phage of Streptococcus pneumoniae.

The abundant presence of temperate phages in the chromosomes of clinical isolates of Streptococcus pneumoniae has been well documented. The genome of MM1, a temperate phage of pneumococcus, has been isolated as a DNA-protein complex. The protein is covalently bound to the DNA, was iodinated in vitro with Na125I, and has an Mr of 22,000. Electron microscopy and enzymatic analyses revealed that the MM1 genome is a linear, circularly permuted, terminally redundant collection of double-stranded DNA molecules packaged via a headful mechanism. The location of the pac site appears to be downstream of the terminase, between orf32 and orf34 of the MM1 genome.

DNA Packaging↗

PubMed1985

EcoK restriction during in vitro packaging of coliphage lambda DNA.

The K restriction system of Escherichia coli works in vitro [Meselson and Yuan, Nature 217 (1968) 1110-1114]. E. coli C lacks the K restriction system. I show that in vitro packaging in standard E. coli K-12-derived systems effects a loss of plaque-former output from K-unmodified lambda DNA relative to K-modified lambda DNA when compared with packaging in the E. coli C-derived system of Rosenberg et al. [Gene 38 (1985) 165-175]. I conclude that the EcoK restriction system is active in standard in vitro packaging systems. EcoK restriction during in vitro packaging could specifically depress recovery of some lambda and cosmid clones of eukaryotic DNA or any other DNA not modified for EcoK restriction.

Bacteriophage lambda↗

PubMed1986

Analysis of deoxyribonucleic acid distribution in noncleaving oocytes from patients undergoing in vitro fertilization.

The purpose of this study was to describe the quantity and distribution of deoxyribonucleic acid (DNA) in oocytes that did not fertilize or did fertilize and failed to cleave, from patients who underwent in vitro fertilization. Patients were selected with at least one cleaving egg, so that the sperm population was known to be fertile, and failure of fertilization or cleavage in the remaining oocytes could be attributed to nonspermatozoan factors. The noncleaving oocytes were classified into five categories, the majority of which (71%) lacked a polar body and any morphologically identifiable nucleus or germinal vesicle. Three general defects were found: failure to replicate the DNA properly; failure to package the DNA properly; and failure to organize the nuclear material properly after sperm penetration. It is concluded that either altered stimulation protocols or altered in vitro maturation conditions are needed to increase the average number of normal embryos available for transfer.

Cleavage Stage, Ovum↗

PubMed2000

Poly(dA.dT) sequences exist as rigid DNA structures in nucleosome-free yeast promoters in vivo.

Poly(dA.dT) sequences (T-tracts) are abundant genomic DNA elements with unusual properties in vitro and an established role in transcriptional regulation of yeast genes. In vitro T-tracts are rigid, contribute to DNA bending, affect assembly in nucleosomes and generate a characteristic pattern of CPDs (cyclobutane pyrimidine dimers) upon irradiation with UV light (UV photofootprint). In eukaryotic cells, where DNA is packaged in chromatin, the DNA structure of T-tracts is unknown. Here we have used in vivo UV photofootprinting and DNA repair by photolyase to investigate the structure and accessibility of T-tracts in yeast promoters (HIS3, URA3 and ILV1). The same characteristic photofootprints were obtained in yeast and in naked DNA, demonstrating that the unusual T-tract structure exists in living cells. Rapid repair of CPDs in the T-tracts demonstrates that these T-tracts were not folded in nucleosomes. Moreover, neither datin, a T-tract binding protein, nor Gcn5p, a histone acetyltransferase involved in nucleosome remodelling, showed an influence on the structure and accessibility of T-tracts. The data support a contribution of this unusual DNA structure to transcriptional regulation.

Acetyltransferases↗

PubMed1993

Characterization of a temperature-sensitive mutant of the UL15 open reading frame of herpes simplex virus 1.

The UL15 gene of herpes simplex virus 1 consists of two exons and is highly conserved among the herpesviruses sequenced to date. Other than its homology to a phage protein involved in the packaging of DNA, nothing is known of its function. This report concerns the isolation of a temperature-sensitive mutant with a mutation mapping in the UL15 open reading frame. Cells infected with the parent, mutant, and rescued viruses all make DNA at the nonpermissive temperature. Direct analyses of the DNA and electron microscopic studies indicate that although viral DNA is made, it is not packaged into capsids present in nuclei. These studies suggest that UL15 may be involved in the packaging of viral DNA.

Animals↗

PubMed2004

Understanding the consequences of epigenetic mechanisms and its effects on transcription in health and disease.

Over the course of thirty-five years of research into the chromosome we are confronted with a number of general conclusions central to chromatin biology, perhaps most pertinent of these is that organization of higher order nucleosome structures play a fundamental role in DNA behavior. Folding and packaging of DNA is linked with a number of processes ranging from transcriptional regulation, genome stability, chromosome segregation, including replication and repair. Intrinsic to nuclear function is chromatin structure and the relationship with epigenetic modification. While we are aware that genetic and epigenetic changes can influence tumorigenesis, it is still unclear what protein components are specified on chromatin or the molecular events that underlie the link between chromosomes and cancer. In recent years, our understanding of DNA methylation with cancer development has revealed some surprising findings with particular emphasis on the antithetical character of hypo- and hyper- methylation of DNA. The molecular mechanism by which methylation is responsible for increased cancer risk has received widespread attention and is relavent to our understanding of genetic stability. The biological significance of other epigenetic modifications such as histone tail changes and active chromatin remodeling are also a major driving force in chromosome research in cancer. In light of recent advances in the understanding of epigenetic changes in disease we have organized a focused review series and discuss the significance of Epigenetics in Normal Control and Deregulation in Cancer. Authors from divergent fields of study have contributed articles that examine chromatin dynamics in cancer, bringing together research interests from a wide variety of fields. In this vein, we discuss the consequences of deregulating epigenetic mechanisms with a viewpoint on transcriptional control, chromatin function and human disease.

Chromatin↗

PubMed1984

Internal motion of DNA in bacteriophages.

We have investigated internal motion of DNA in bacteriophages by measuring fluorescence anisotropy decays of intercalated ethidium. The results showed large suppression of the internal motion of the inner DNA; the interhelix interaction of the DNA in the phage head is considered to enhance the effective viscosity of the DNA rod and to restrict the angle of the internal motion. Considering that the observed internal motion arises mainly from torsional motion of the DNA, we have calculated the movable angles of the torsional motion (the standard deviation of the torsional motion) of the DNA in the phage heads. The magnitude of the calculated movable angles indicates the extent of suppression of the DNA movement in the phage head; in lambda wild type phage, the DNA is packed most rigidly in the head and the motion is found to be restricted most severely. In a deletion mutant of lambda phage, whose inner DNA content is deficient by 17.6%, steric hindrance from the interhelix DNA interaction is decreased, and the DNA can move more easily. In T4 wild type phage, although the extent of condensation of the inner DNA is the same as that in lambda wild type phage, the DNA was fairly mobile. The presence of glucosylated hydroxymethylcytosine is suggested to influence the rigidity of the inner DNA or packaging mode of the DNA in the T4 head.

Animals↗

PubMed1996

The simian virus 40 packaging signal ses is composed of redundant DNA elements which are partly interchangeable.

Using the experimental system of simian virus 40 (SV40) pseudovirions we have previously shown that SV40 requires a specific DNA element for packaging, ses, which was mapped to the SV40 regulatory region. ses was previously found to play a role in facilitating the nucleosomal rearrangement required for chromatin condensation and viral packaging. Here, the fine structure of ses was investigated by genetic studies. Analyses of ses+ revertants indicated that in order to function, ses must be present in close proximity to the origin of replication (ori), supporting a role in the regulation of the viral life cycle. Fine dissection of ses was performed using a series of plasmids carrying mutations and deletions in this region. The results suggest that multiple DNA elements participate in the SV40 packaging process, including the GC-boxes and elements derived from the enhancer. The elements are redundant, and they can function in various combinations. Packaging efficiency correlated with the number of GC-boxes, known to bind Sp1. In addition, AP-2 binding elements appeared to more important than others. These findings were supported by experiments which showed that packaging was significantly enhanced by adding AP-2 binding sites to plasmids with large deletions and lacking those sites. The results imply that binding of Sp1 and/or AP-2 may participate in the packaging process.

Animals↗

PubMed2006

Histone post-translational modifications and the response to DNA double-strand breaks.

The packaging of DNA into chromatin creates a number of significant barriers to the detection of DNA lesions and their timely and accurate repair. Eukaryotic cells have evolved a number of enzymes that modulate chromatin structure and facilitate DNA repair. Recent research illustrates how nucleosome remodelling enzymes cooperate with both DNA-damage-inducible and constitutive histone modifications to promote many facets of the cellular response to DNA damage.

Acetylation↗

PubMed1980

Recognition of specific DNA sequences in eukaryotic chromosomes.

The packaging of DNA into chromatin probably places certain restrictions on how specific DNA sequences can be recognized by DNA sequence specific recognition proteins (SRP). Several unique features of this type of interaction are discussed. Specifically, as a consequence of the coiling of the DNA about a histone core, it is proposed that DNA recognition sites will be compound and that each element of the compound recognition site will be about 10 - 20 b.p. in length and distributed at approximately 80 b.p. intervals--the periodicity of the DNA wrapping around the nucleosome.

Base Sequence↗

PubMed1990

Adenovirus type 5 packaging domain is composed of a repeated element that is functionally redundant.

Previous analyses have demonstrated that adenovirus DNA is packaged into virions in vivo in a polar, left-to-right fashion. The packaging of viral DNA is dependent on cis-acting elements at the left end of the genome. In this report, we describe a genetic analysis of the sequences that are required for efficient packaging of adenovirus type 5 (Ad5) DNA. Our results demonstrate that the Ad5 packaging domain (nucleotides 194 to 358) is composed of at least five distinct elements that are functionally redundant. An AT-rich repeated sequence motif, the A repeat, is located in four of five of these regions; the fifth region is also AT rich. The efficiency of viral packaging depends on the number of individual A repeats that are present in the viral genome. The deletion of the entire packaging domain resulted in the loss of virus viability. A virus that contains a multimerized oligonucleotide corresponding to A repeat II in place of the packaging domain could package viral DNA, although with reduced efficiency compared with that of the wild-type virus. Our results also suggest that the spacing of specific sequences at the left end of the Ad5 genome are important for enhancer region function in vivo.

Adenoviruses, Human↗

PubMed2001

Comparative evaluation of three commercial software packages for analysis of DNA polymorphism patterns.

OBJECTIVE: In the present study we have compared three commercial software packages, GelCompar, Molecular Analyst Fingerprinting, and BioImage, to determine if the results generated by the programs were comparable and correlated adequately with visual interpretation of electrophoretic gels, in the analysis of several well characterized incidents of infections. METHODS: Infections caused by Pseudomonas aeruginosa, Candida dubliniensis, C. albicans, and serotypes of Salmonella were characterized by restriction endonuclease analysis, macrorestriction analysis of genomic DNA with pulsed-field gel electrophoresis, and random amplified polymorphic DNA. The genotypes were visually detected based on band presence or absence in the different gels. The similarity values of DNA profiles were computed using Dice coefficient and were presented in dendrograms by UPGMA. The concordance or agreement between the number of genotypes obtained and their clustering, using the computerized programs, was determined. RESULTS: In general, agreement in number of genotypes obtained visually and by using the commercial DNA analysis software was achieved, but discrepancies were also denoted between the systems. The concordance between the visual and the computerized analysis ranged from 72% to 100%. CONCLUSION: In our experience, although the programs evaluated in the present study performed acceptably well, such programs may be used as an aid in the analysis of complex banding patterns, and they do not provide an indisputably correct analysis in genotype definition.

Animals↗