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PubMed1985

Expression of an immediate early polypeptide and activation of a viral origin of DNA replication in cells containing a fragment of herpes simplex virus DNA.

A thymidine kinase cotransformation procedure has been used to introduce the sequences encoding the herpes simplex virus type 1 (HSV-1) immediate early protein, Vmw175, into permissive cells either in the presence or the absence of the adjacent origin of viral DNA replication. Cells transformed by either origin-plus or origin-minus DNA were capable of expressing functional Vmw175 as indicated by their ability to complement the growth at the nonpermissive temperature of an HSV-1 mutant, ts K, containing a temperature-sensitive lesion in the Vmw175 gene. A proportion of the virus yield from cells transformed with the origin-plus, but not the origin-minus, plasmid exhibited a ts+ phenotype. The generation of ts+ virus correlated with an amplification of input plasmid DNA sequences which occurred following superinfection, suggesting that recombination between the ts mutant and the amplified viral DNA sequences had taken place. Encapsidation of the amplified DNA sequences was also detected, suggesting that in addition to a functional origin of replication and Vmw175 gene the transformed cells also retain the viral DNA packaging signals.

Capsid↗
PubMed2002

Sequential model of phage PRD1 DNA delivery: active involvement of the viral membrane.

DNA translocation across the barriers of recipient cells is not well understood. Viral DNA delivery mechanisms offer an opportunity to obtain useful information in systems in which the process can be arrested to a number of stages. PRD1 is an icosahedral double-stranded (ds)DNA bacterial virus with an internal membrane. It is an atypical dsDNA phage, as any of the vertex spikes can be used for receptor recognition. In this report, we dissect the PRD1 DNA entry into a number of steps: (i) outer membrane (OM) penetration; (ii) peptidoglycan digestion; (iii) cytoplasmic membrane (CM) penetration; and (iv) DNA translocation. We present a model for PRD1 DNA entry proposing that the initial stage of entry is powered by the pressure build-up during DNA packaging. The viral protein P11 is shown to function as the first DNA delivery protein needed to penetrate the OM. We also report a DNA translocation machinery composed of at least three viral integral membrane proteins, P14, P18 and P32.

Bacteriophage PRD1↗
PubMed2001

Distinct and separate roles for herpesvirus-conserved UL97 kinase in cytomegalovirus DNA synthesis and encapsidation.

The human cytomegalovirus UL97 kinase, an important target of antiviral therapy, has an impact on at least two distinct phases of viral replication. Compared with wild-type virus, the UL97 deletion mutant exhibits an early replication defect that reduces DNA accumulation by 4- to 6-fold, as well as a late capsid maturation defect responsible for most of the observed 100- to 1000-fold reduction in replication. Block-release experiments with the antiviral 2-bromo-5,6-dichloro-1-(beta-D-ribofuranosyl)-benzimidazole revealed an important role for UL97 kinase in capsid assembly. Although cleavage of concatemeric DNA intermediates to unit-length genomes remained unaffected, progeny mutant virus maturation was delayed, with accumulation of progeny at significantly reduced levels compared with wild type after release of this block. Transmission electron microscopy confirmed the aberrant accumulation of empty A-like capsids containing neither viral DNA nor an internal scaffold structure, consistent with a failure to stably package DNA in mutant virus-infected cells. The function of UL97 in DNA synthesis as well as capsid assembly suggests that protein phosphorylation mediated by this herpesvirus-conserved kinase increases the efficiency of these two distinct phases of virus replication.

Capsid↗
PubMed2001

ATPase activity of the terminase subunit pUL56 of human cytomegalovirus.

Herpesviral DNA packaging is a complex process resulting in unit-length genomes packed into preformed procapsids. This process is believed to be mediated by two packaging proteins, the terminase subunits. In the case of double-stranded DNA bacteriophages, the translocation of DNA was shown to be an energy-dependent process associated with an ATPase activity of the large terminase subunit. In the case of human cytomegalovirus it was not known which protein has the ability to hydrolyze ATP. In this study we expressed human cytomegalovirus terminase subunits, pUL89 and the carboxyl-terminal half of pUL56, as GST fusion proteins and purified these by affinity chromatography. ATPase assays demonstrated that the enzymatic activity is exclusively associated with pUL56. The characterization of the ATP hydrolysis showed that the enzymatic reaction is a fast process, whereas the spontaneous ATP decay followed slow kinetics. Interestingly, although pUL89 did not show any ATPase activity, it was capable of enhancing the UL56-associated ATP hydrolysis. Furthermore, a specific association of in vitro translated pUL89 with the carboxyl-terminal half of GST-UL56C was detected. This interaction was confirmed by co-immunoprecipitations of infected cells. Our results clearly demonstrated that (i) both terminase subunits interact with each other and (ii) the subunit pUL56 has an ATPase activity.

Adenosine Triphosphatases↗
PubMed2002

Unique condensation patterns of triplex DNA: physical aspects and physiological implications.

Triple-stranded DNA structures can be formed in living cells, either by native DNA sequences or following the application of antigene strategies, in which triplex-forming oligonucleotides are targeted to the nucleus. Recent studies imply that triplex motifs may play a role in DNA transcription, recombination and condensation processes in vivo. Here we show that very short triple-stranded DNA motifs, but not double-stranded segments of a comparable length, self-assemble into highly condensed and ordered structures. The condensation process, studied by circular dichroism and polarized-light microscopy, occurs under conditions that mimic cellular environments in terms of ionic strength, ionic composition and crowding. We argue that the unique tendency of triplex DNA structures to self-assemble, a priori unexpected in light of the very short length and the large charge density of these motifs, reflects the presence of strong attractive interactions that result from enhanced ion correlations. The results provide, as such, a direct experimental link between charge density, attractive interactions between like-charge polymers and DNA packaging. Moreover, the observations strongly support the notion that triple-stranded DNA motifs may be involved in the regulation of chromosome organization in living cells.

Chromosomes↗
PubMed1989

Movement of multiple DNA units between Myxococcus xanthus cells.

Myxococcus xanthus YS produces particles (Mx alpha particles) that transmit genetic information between cells. Mx alpha particles might be viruses, although no host able to sustain lytic growth of Mx alpha has been discovered. The particles could be detected by their ability to transduce a Tn5 transposon tag to recipient bacteria. DNA from purified particles hybridized to a limited number of DNA restriction fragments of strain YS, suggesting that Mx alpha particles contain only specific DNA sequences. A set of Tn5 insertions residing in the transducible region provided genetic markers for cloning cellular DNA packaged by Mx alpha. A map of this region showed that transducible DNA comprised multiple units of approximately 80 kilobases each. Individual units share DNA homology but are divergent in the location of restriction sites. Other wild-type isolates of Myxococcus species contained DNA sequences with homology to Mx alpha DNA, indicating that Mx alpha DNA is widespread in nature. Experiments on the transfer of Mx alpha DNA in strain YS suggested that DNA transfer is enhanced during the developmental cycle.

Blotting, Southern↗
PubMed1998

Detection of N6-methyladenine in GATC sequences of Selenomonas ruminantium.

The presence of N6-methyladenine in GATC sequences in DNA of Selenomonas ruminantium was investigated using sensitive methylation discriminating isochizomeric restriction enzymes analysis. Methylated adenine was detected in 8 out of 18 tested strains belonging to the subsp. lactilytica of S. ruminantium. No corresponding restriction activity was detected in three tested strains. No GATC methylation was detected in 3 analysed S. ruminantium subsp. ruminantium strains. Sustainable progress was achieved in the molecular biology of ruminal microorganisms in the last decade. Many different genes acting in the cell wall degradation were cloned and characterized. As practically all cloning experiments were done in Escherichia coli cells, there is a lack of data about regulation of gene(s) expression in the natural hosts. However, much better understanding of molecular genetics of ruminal bacteria is required for improving rumen functions by genetic modifications of rumen bacteria. DNA methylation is main mechanism of the control of gene expression in eukaryotes. In prokaryotes, DNA methylation influences wide variety of important cellular functions as accessibility of DNA to digestion by restriction endonucleases, control of replication initiation, transposition, phage DNA packaging, including positive and negative regulation of gene expression. Most of the DNA methyltransferases; enzymes which facilitate methylation; identified in prokaryotes are part of restriction modification systems (WILSON and MURRAY 1991. Another class of methyltransferase are independent methylases like Dam and Dcm in Escherichia coli. Dam methylase recognizes the sequence GATC and methylates adenine at N6 position (BARRAS and MARINUS 1989). The methylation is the only documented case of prokaryotic methylation involved in the regulation of cellular process (NOYER-WIEDNER and TRAUTNER 1993). Screening of large number of bacteria have detected the presence of Dam methylation in cyanobacteria as well as in the group of related families of Enterobacteriaceae (which includes E. coli), Parvobacteriaceae and Vibrionaceae. Methylated GATC sequences have been found in several other bacterial species (NOYER-WIEDNER and TRAUTNER 1993), however, it could be assumed that some of these methylations are due to the presence of restriction modification systems. There is no data about the presence of Dam methylation neither in Selenomonas ruminantium nor in any other ruminal bacteria. Bacteria of S. ruminantium species are known to contain frequently restriction modification systems. Several restriction endonucleases were characterized from these bacteria (VANAT et al., 1993, PRISTAS et al. 1994, 1995). During characterization of modification activities associated with these endonucleases we have detected the modification of adenine in GATC sequences of DNA of this species.

Adenine↗
PubMed2002

Inhibition of herpes simplex virus replication by WAY-150138: assembly of capsids depleted of the portal and terminase proteins involved in DNA encapsidation.

Studies were carried out to examine the mechanism of action of WAY-150138, a member of a novel group of thiourea compounds recently shown to inhibit replication of herpes simplex virus type 1 (HSV-1). Previous studies have shown that the drug acts by preventing DNA encapsidation and that resistant mutants map to U(L)6, the gene encoding the protein subunit of the portal complex through which DNA enters the capsid. We tested the idea that WAY-150138 acts by preventing the incorporation of DNA-packaging proteins into capsids as they are assembled. Capsids were isolated from HSV-1-infected, drug-treated cells and examined by Western immunoblotting for the presence of two packaging proteins, the portal subunit (U(L)6) and a candidate terminase subunit (U(L)15). The results showed that both proteins were depleted in the capsids, suggesting that WAY-150138 antagonizes DNA encapsidation by depriving capsids of packaging proteins during the assembly process.

Animals↗
PubMed2004

Comparative genomics of the FtsK-HerA superfamily of pumping ATPases: implications for the origins of chromosome segregation, cell division and viral capsid packaging.

Recently, it has been shown that a predicted P-loop ATPase (the HerA or MlaA protein), which is highly conserved in archaea and also present in many bacteria but absent in eukaryotes, has a bidirectional helicase activity and forms hexameric rings similar to those described for the TrwB ATPase. In this study, the FtsK-HerA superfamily of P-loop ATPases, in which the HerA clade comprises one of the major branches, is analyzed in detail. We show that, in addition to the FtsK and HerA clades, this superfamily includes several families of characterized or predicted ATPases which are predominantly involved in extrusion of DNA and peptides through membrane pores. The DNA-packaging ATPases of various bacteriophages and eukaryotic double-stranded DNA viruses also belong to the FtsK-HerA superfamily. The FtsK protein is the essential bacterial ATPase that is responsible for the correct segregation of daughter chromosomes during cell division. The structural and evolutionary relationship between HerA and FtsK and the nearly perfect complementarity of their phyletic distributions suggest that HerA similarly mediates DNA pumping into the progeny cells during archaeal cell division. It appears likely that the HerA and FtsK families diverged concomitantly with the archaeal-bacterial division and that the last universal common ancestor of modern life forms had an ancestral DNA-pumping ATPase that gave rise to these families. Furthermore, the relationship of these cellular proteins with the packaging ATPases of diverse DNA viruses suggests that a common DNA pumping mechanism might be operational in both cellular and viral genome segregation. The herA gene forms a highly conserved operon with the gene for the NurA nuclease and, in many archaea, also with the orthologs of eukaryotic double-strand break repair proteins MRE11 and Rad50. HerA is predicted to function in a complex with these proteins in DNA pumping and repair of double-stranded breaks introduced during this process and, possibly, also during DNA replication. Extensive comparative analysis of the 'genomic context' combined with in-depth sequence analysis led to the prediction of numerous previously unnoticed nucleases of the NurA superfamily, including a specific version that is likely to be the endonuclease component of a novel restriction-modification system. This analysis also led to the identification of previously uncharacterized nucleases, such as a novel predicted nuclease of the Sir2-type Rossmann fold, and phosphatases of the HAD superfamily that are likely to function as partners of the FtsK-HerA superfamily ATPases.

Adenosine Triphosphatases↗
PubMed1999

Domain architecture of the bacteriophage phi29 connector protein.

Viral connectors are essential components of the DNA packaging machinery. They interact with nucleic acids and other viral components to translocate DNA inside the viral head. We have attempted to locate the different structural and functional domains of the phage Phi29 connector using a combination of approaches to generate different antigenic probes. Complexes of native connectors with either monoclonal or monospecific antibodies were studied by immunoelectron microscopy and image averaging methods. The data were merged in a model of the connector domain structure at 2-3 nm resolution. This epitope mapping provides a general outline of the folding architecture of the connector polypeptide, following a complicated threading that places the amino and carboxyl-terminals in close alignment in the narrower domain at 2-3 nm from the top of the connector. The appendages are built up by a long and highly immunogenic sequence (amino acid residues 153 to 206). The RNA binding domain forms part of the top of the narrow conical area of the connector, a flexible region that undergoes structural changes during viral morphogenesis. The DNA binding domain is located not far away, 2-3 nm below, in the outer side of the narrow conical part. The precise location of the functional domains of the connector, as well as their relative positions provide the first experimental framework for understanding the connector function.

Amino Acid Sequence↗
PubMedDate not supplied

The interaction of DNA with bacteriophage phi 29 connector: a study by AFM and TEM.

The connector of bacteriophage phi 29 is involved in DNA packaging during viral morphogenesis and we have studied its in vitro binding to DNA using either linear or circular DNA. The protein-DNA complexes have been analyzed by transmission electron microscopy (TEM) and by atomic force microscopy (AFM) of samples directly deposited on mica. TEM showed the presence of a specific binding due to the interaction of the protein with the free ends of the DNA. The study of these samples by AFM showed two major types of morphologies: The interaction of the connector with circular DNA revealed that the strands of DNA that enter and exit the protein complex form an angle with a mean value of 132 degrees. Nevertheless, when the connector was incubated with linear DNA (and later circularized), there was an additional bend angle of about 168 degrees. Further morphological analysis of the latter samples by AFM revealed a structure of the protein-DNA complex consistent with the DNA traversing the connector, probably through the inner channel. On the other hand, images from the samples obtained by incubation of the connector with circular DNa were consistent with an interaction of the DNA with the outer side of the connector.

Bacillus Phages↗
PubMed1996

Mapping functional domains in AcMNPV pp31.

Autographa californica nuclear polyhedrosis virus (AcMNPV) replicates in the nucleus and produces a viral-modified form of the nuclear matrix called the virogenic stroma. The virogenic stroma is the site of viral DNA packaging and nucleocapsid assembly and is thought to be the site of viral DNA replication and RNA transcription. AcMNPV encodes a phosphoprotein, pp31, which localizes to the nucleus of uninfected insect cells and to the virogenic stroma of infected insect cells. pp31 has DNA binding activity and has been identified as a late expression factor. Thus, the intracellular location of pp31, its DNA binding activity, and its identification as a late transcription factor suggest that it participates in replicative events that occur in the virogenic stroma during AcMNPV infection. The purpose of this study was to map the pp31 domains needed for nuclear localization, virogenic stroma localization, and DNA binding. We focused on four basic amino acid regions (BRs 1-4) and used site-directed mutagenesis and gene fusion techniques to probe their functions. The amino-terminal basic region (BR1) was most important for nuclear localization of pp31 in uninfected cells. Three of the four BRs were needed to efficiently localize pp31 to the nucleus and virogenic stroma in infected cells. BR3 was identified as the DNA binding domain of pp31. These data indicated that BR1, BR3, and BR4 are important functional or multifunctional domains within the AcMNPV pp31 protein.

Animals↗
PubMed1990

A binding protein (p82 protein) recognizes specifically the curved heterochromatic DNA in Artemia franciscana.

DNA bending has been suggested to play a role in the regulation of gene expression, initiation of DNA replication, site specific recombination and DNA packaging. In Artemia franciscana (Phillopoda anostraca) cells we have revealed that an AluI DNA family of repeats, 113-bp in length, is the major component of the constitutive heterochromatin found in the species. By analysis of cloned oligomeric (monomer to hexamer) heterochromatic fragments and electrophoretic experiments we verified that the repetitive DNA shows a stable curvature that confers a solenoidal geometry to the double helix. Using the cloned monomeric fragment, as molecular probe, we describe the detection in an A. franciscana cell extract of a protein of 82 kDa (p82) that preferentially binds to heterochromatic DNA. This protein, purified of the other DNA binding proteins present in the crude cell extract, shows a greater affinity with the tandem copies of the AluI DNA fragment than with the monomer sequence. The binding of p82 protein to heterochromatic DNA is also drastically reduced in the presence of the antibiotic distamycin A, suggesting a role of the DNA curvature in the formation of the nucleoproteic complex.

Animals↗
PubMed1980

Nuclear magnetic resonance of the filamentous bacteriophage fd.

The filamentous bacteriophage fd and its major coat protein are being studied by nuclear magnetic resonance (NMR) spectroscopy. 31P NMR shows that the chemical shielding tensor of the DNA phosphates of fd in solution is only slightly reduced in magnitude by motional averaging, indicating that DNA-protein interactions substantially immobilize the DNA packaged in the virus. There is no evidence of chemical interactions between the DNA backbone and the coat protein, since experiments on solid virus show the 31P resonances to have the same principle elements of its chemical shielding tensor as DNA. 1H and 13C NMR spectra of fd virus in solution indicate that the coat proteins are held rigidly in the structure except for some aliphatic side chains that undergo relatively rapid rotations. The presence of limited mobility in the viral coat proteins is substantiated by finding large quadrupole splittings in 2H NMR of deuterium labeled virions. The structure of the coat protein in a lipid environment differs significantly from that found for the assembled virus. Data from 1H and 13C NMR chemical shifts, amide proton exchange rates, and 13C relaxation measurements show that the coat protein in sodium dodecyl sulfate micelles has a native folded structure that varies from that of a typical globular protein or the coat protein in the virus by having a partially flexible backbone and some rapidly rotating aromatic rings.

Bacterial Proteins↗
PubMed2004

Partial B-to-A DNA transition upon minor groove binding of protein Sac7d monitored by Raman spectroscopy.

Members of the Sso7d/Sac7d protein family and other related proteins are believed to play an important role in DNA packaging and maintenance in archeons. Sso7d/Sac7d are small, abundant, basic, and nonspecific DNA-binding proteins of the hyperthermophilic archeon Sulfolobus. Structures of several complexes of Sso7d/Sac7d with DNA octamers are known. These structures are characterized by sequence unspecific minor groove binding of the proteins and sharp kinking of the double helix. Corresponding Raman vibrational signatures have been identified in this study. A Raman spectroscopic analysis of Sac7d binding to the oligonucleotide decamer d(GAGGCGCCTC)(2) reveals large conformational perturbations in the DNA structure upon complex formation. Perturbed Raman bands are associated with the vibrational modes of the sugar phosphate backbone and frequency shifts of bands assigned to nucleoside vibrations. Large changes in the DNA backbone and partial B- to A-form DNA transitions are indicated that are closely associated with C2'-endo/anti to C3'-endo/anti conversion of the deoxyadenosyl moiety upon Sac7d binding. The major spectral feature of Sac7d binding is kinking of the DNA. Raman markers of minor groove binding do not largely contribute to spectral differences; however, clear indications for minor groove binding come from G-N2 and G-N3 signals that are supported by Trp24 features. Trp24 is the only tryptophan present in Sac7d and binds to guanine N3, as has been demonstrated clearly in X-ray structures of Sac7d-DNA complexes. No changes of the Sac7d secondary structure have been detected upon DNA binding.

Amino Acids↗
PubMed2004

Characterization of nonspecific protein-DNA interactions by 1H paramagnetic relaxation enhancement.

Nonspecific protein-DNA interactions play an important role in a variety of contexts related to DNA packaging, nucleoprotein complex formation, and gene regulation. Biophysical characterization of nonspecific protein-DNA interactions at the atomic level poses significant challenges owing to the dynamic nature of such complexes. Although NMR spectroscopy represents a powerful tool for the analysis of dynamic systems, conventional NMR techniques have provided little information on nonspecific protein-DNA interactions. We show that intermolecular (1)H paramagnetic relaxation enhancement (PRE) arising from Mn(2+) chelated to an EDTA-group covalently attached to a thymine base (dT-EDTA-Mn(2+)) in DNA provides a unique approach for probing the global dynamics and equilibrium distribution of nonspecific protein-DNA interactions. For nonspecific DNA binding, similar intermolecular (1)H-PRE profiles are observed on the (1)H resonances of the bound protein when dT-EDTA-Mn(2+) is located at either end of a DNA oligonucleotide duplex. We demonstrate the applicability of this approach to HMG-box proteins and contrast the results obtained for nonspecific DNA binding of the A-box of HMGB-1 (HMGB-1A) with sequence-specific DNA binding of the related SRY protein. Intermolecular (1)H-PRE data demonstrate unambiguously that HMGB-1A binds to multiple sites in multiple orientations even on a DNA fragment as short as 14 base pairs. Combining the (1)H-PRE data with the crystal structure of the HMGB-1 A-box/cisplatin-modified DNA complex allows one to obtain a semiquantitative estimate of the equilibrium populations at the various sites.

Amino Acid Sequence↗
PubMed1992

Radical effects on mutation spectra in lambda phage.

Mutations in the lambda repressor gene cI (710 bp) were induced by 60Co-gamma radiation in dissolved lambda phage DNA. After in vitro DNA packaging to lambda phage particles (pack phage) and phenotypic expression of the mutants, DNA was sequenced directly. Two-thirds of mutations were located in the amino terminus region of the gene without any signs of hotspots. Changes consisted of (+1) insertions (25%) and base substitutions (75%). Transitions were exclusively G/C to A/T. Transversions were mostly G/C to C/G and few G/C to T/A. We did not find A/T to T/A transversions, A/T to G/C transitions, deletions and gross rearrangements. In most of the base substitutions a pre-existing base pair had been replaced by an A/T pair; this might come from 'non-instructional sites' like abasic sites. Several mechanisms for base substitutions are considered.

Bacteriophage lambda↗
PubMed1981

Genetic recombination of bacteriophage T7 in vivo studied by use of a simple physical assay.

A new physical method was developed to assay genetic recombination of phage T7 in vivo. The assay utilized T7 mutants that carry unique restriction sites and was based on the detection of a new restriction fragment generated by recombination. Using this assay, we reexamined the genetic requirements for recombination of T7 DNA. Our results were in total agreement with previous findings in that recombination required the products of genes 3 (endonuclease), 4 (primase), 5 (DNA polymerase), and 6 (exonuclease). Recombination was found to be independent of DNA ligase and DNA packaging and maturation functions.

DNA Primase↗