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PubMed1978

The compaction of DNA helices into either continuous supercoils or folded-fiber rods and toroids.

We have investigated by electron microscopy the mechanism of DNA compaction and have found that the double helix has the intrinsic potential to direct its own packaging into two distinctly different and mutally exclusive modes. The mode of DNA packaging is determined by the electrostatic charge density and water activity of the immediate microenvironment of the helix. The two basic structures formed by both linear and covalently closed-circular DNA are: a left-handed supercoil characteristic of minimally charge-shielded DNA, and a smooth rod characteristic of fully charge-shielded DNA. We propose that in the supercoil, the double helix is overwound (increased turn-angle), while in the rod, the helix is folded back and forth on itself. Variation of these two basic structures are the beaded fiber of DNA obtained with partially charge-shielded DNA and the toroid formed by the bending of the DNA rod and fusion of its ends in the presence of certain cations. We compare the DNA packaging inside these in vitro generated structures to DNA packaging in chromatin and viral capsids, and conclude that the packaging of DNA brought about by the use of salts and alcohol closely mimics the packaging behavior of the DNA in vivo, where it is usually complexed with histones or polyamines.

Bacteriophages↗

PubMed1998

In vitro packaging of adeno-associated virus DNA.

We have developed an in vitro procedure for packaging of recombinant adeno-associated virus (AAV). By using AAV replicative-form DNA as the substrate, it is possible to synthesize an infectious AAV particle in vitro that can be used to transfer a marker gene to mammalian cells. The packaging procedure requires the presence of both the AAV Rep and capsid proteins. Two kinds of in vitro products can be formed which facilitate DNA transfer. Both are resistant to heat and have a density in cesium chloride gradients that is indistinguishable from that of the in vivo-synthesized wild-type virus. This indicates that the particles formed have the appropriate protein-to-DNA ratio and a structure that shares the heat resistance of mature AAV particles. The two types of particles can be distinguished by their sensitivity to chloroform and DNase I treatment. The chloroform-resistant product is, by several criteria, an authentic AAV particle. In addition to having the correct density and being resistant to treatment with chloroform, DNase I, and heat, this particle is efficiently synthesized only if the AAV genome contains intact terminal repeats, which are known to be required for AAV packaging. It is also precipitated by a monoclonal antibody that recognizes mature virus particles but not bound by an antibody that recognizes monomeric or denatured capsid proteins. The chloroform-resistant species is not made when aphidicolin is present in the reaction mixture, suggesting that active DNA replication is required for in vitro packaging. In contrast, the chloroform-sensitive product has several features that suggest it is an incompletely assembled virus particle. It is sensitive to DNase I, does not require the presence of AAV terminal repeats, and is capable of transferring DNA that is theoretically too large to package. Sucrose gradient centrifugation of the in vitro-synthesized products reveals that the particles have sedimentation values between 60S and 110S, which is consistent with partially assembled and mature AAV particles. The in vitro packaging procedure should be useful for studying the mechanism by which a human icosahedral DNA virus particle is assembled, and it may be useful for producing recombinant AAV for gene therapy. The chloroform-sensitive particle may also be useful for transferring DNA that is too large to be packaged in mature recombinant AAV.

Adenosine Triphosphate↗

PubMed1991

Isolation and characterization of mutations in the bacteriophage lambda terminase genes.

The terminase enzyme of bacteriophage lambda is a hetero-oligomeric protein which catalyzes the site-specific endonucleolytic cleavage of lambda DNA and its packaging into phage proheads; it is composed of the products of the lambda Nul and A genes. We have developed a simple method to select mutations in the terminase genes carried on a high-copy-number plasmid, based on the ability of wild-type terminase to kill recA strains of Escherichia coli. Sixty-three different spontaneous mutations and 13 linker insertion mutations were isolated by this method and analyzed. Extracts of cells transformed by mutant plasmids displayed variable degrees of reduction in the activity of one or both terminase subunits as assayed by in vitro lambda DNA packaging. A method of genetically mapping plasmid-borne mutations in the A gene by measuring their ability to rescue various lambda Aam phages showed that the A mutations were fairly evenly distributed across the gene. Mutant A genes were also subcloned into overproducing plasmid constructs, and it was determined that more than half of them directed the synthesis of normal amounts of full-length A protein. Three of the A gene mutants displayed dramatically reduced in vitro packaging activity only when immature (uncut) lambda DNA was used as the substrate; therefore, these mutations may lie in the endonuclease domain of terminase. Interestingly, the putative endonuclease mutations mapped in two distinct locations in the A gene separated by a least 400 bp.

Bacteriophage lambda↗

PubMed1987

Recognition and cleavage of the bacteriophage P1 packaging site (pac). I. Differential processing of the cleaved ends in vivo.

The packaging of bacteriophage P1 DNA into viral capsids is initiated at a specific DNA site called pac. During packaging, that site is cleaved and at least one of the resulting ends is encapsidated into a P1 virion. We show here that pac is located on a 620 base-pair fragment of P1 DNA (EcoRI-20). When that fragment is inserted into the chromosome of cells that are then infected with P1, packaging of host DNA into phage particles is initiated at pac and proceeds down the chromosome, unidirectionally, for about five to ten P1 "headfuls" (about 5 X 10(5) to 10 X 10(5) bases of DNA). Using an assay for pac cleavage that does not depend on DNA packaging, we have identified a set of five amber mutations that are mapped adjacent to pac, and that define a gene (gene 9) essential for pac cleavage. Amber mutations that are located in genes necessary for viral capsid formation (genes 4, 8 and 23), or in a gene necessary for "late" protein synthesis (gene 10), do not affect pac cleavage. The latter result suggests that the synthesis of the pac cleavage protein is not regulated co-ordinately with other phage morphogenesis proteins. The products of pac cleavage were analyzed using two different DNA substrates. In one case, a single copy of pac was placed in the chromosome of P1-sensitive cells. When those cells were infected with P1, we could detect the cleavage of as much as 70% of the pac-containing DNA. The pac end destined to be packaged in the virion was detected five to 20 times more efficiently than was the other end. Since this result is obtained whether or not the infecting P1 phage can encapsidate the cut pac site, the differential detection of pac ends is not simply a consequence of one end being packaged and the other not. In a second case, pac was located in cells on a small (5 X 10(3) bases) multicopy plasmid. When those cells were infected with P1, neither pac end was detected efficiently after P1 infection, unless the cells carried a recBCD- mutation. In recBCD- cells, the results with plasmid-pac substrates were similar to those obtained with chromosomally integrated pac substrates. We interpret these results to mean that, following pac cleavage, the end destined to be packaged is protected from cellular nucleases while the other end is degraded by the action of at least two nucleases, one of which is the product of the host recBCD gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Bacteriophages↗

PubMed1986

In vitro packaging of heteroduplex bacteriophage T7 DNA: evidence for repair of mismatched bases.

Heteroduplex DNA molecules that were wild type or contained combinations of amber, missense, and temperature-sensitive mutations were prepared from bacteriophage T7. These DNA molecules were then encapsulated in in vitro packaging reactions so as to produce infective T7 phage. The genotypes of the phage were examined to determine the degree to which mismatched base pairs in the heteroduplex had been corrected. The data show that conversion of the mismatches took place either during in vitro packaging or immediately after infection of either an Escherichia coli or Shigella sonnei host. The mode of mismatch conversion observed in these experiments was independent of the host mutH, mutL, mutS, and uvrD genes. There was no significant amount of discrimination between markers on either of the two complementary strands. The observed frequency of conversion of a mismatch depended on the genetic marker being monitored and on experimental conditions but was generally in the range between 5 and 30%.

DNA Repair↗

PubMed2006

Bacteriophage lambda gpNu1 and Escherichia coli IHF proteins cooperatively bind and bend viral DNA: implications for the assembly of a genome-packaging motor.

Terminase enzymes are common to both prokaryotic and eukaryotic double-stranded DNA viruses and are responsible for packaging viral DNA into the confines of an empty procapsid shell. In all known cases, the holoenzymes are heteroligomers composed of a large subunit that possesses the catalytic activities required for genome packaging and a small subunit that is responsible for specific recognition of viral DNA. In bacteriophage lambda, the DNA recognition protein is gpNu1. The gpNu1 subunit interacts with multiple recognition elements within cos, the packaging initiation site in viral DNA, to site-specifically assemble the packaging machinery. Motor assembly is modulated by the Escherichia coli integration host factor protein (IHF), which binds to a consensus sequence also located within cos. On the basis of a variety of biochemical data and the recently solved NMR structure of the DNA binding domain of gpNu1, we proposed a novel DNA binding mode that predicts significant bending of duplex DNA by gpNu1 (de Beer et al. (2002) Mol. Cell 9, 981-991). We further proposed that gpNu1 and IHF cooperatively bind and bend viral DNA to regulate the assembly of the packaging motor. Here, we characterize cooperative gpNu1 and IHF binding to the cos site in lambda DNA using a quantitative electrophoretic mobility shift (EMS) assay. These studies provide direct experimental support for the long presumed cooperative assembly of gpNu1 and IHF at the cos sequence of lambda DNA. Further, circular permutation experiments demonstrate that the viral and host proteins each introduce a strong bend in cos-containing DNA, but not nonspecific DNA substrates. Thus, specific recognition of viral DNA by the packaging apparatus is mediated by both DNA sequence information and by structural alteration of the duplex. The relevance of these results with respect to the assembly of a viral DNA-packaging motor is discussed.

Bacteriophage lambda↗

PubMed1997

Magnesium-induced conformational change of packaging RNA for procapsid recognition and binding during phage phi29 DNA encapsidation.

Bacteriophage phi29 is typical of double-stranded DNA viruses in that its genome is packaged into a preformed procapsid during maturation. An intriguing feature of phi29 assembly is that a virus-encoded RNA (pRNA) is required for the packaging of its genomic DNA. Psoralen cross-linking, primer extension, and T1 RNase partial digestion revealed that pRNA had at least two conformations; one was able to bind procapsids, and the other was not. In the presence of Mg2+, one stretch of pRNA, consisting of bases 31 to 35, was confirmed to be proximal to base 69, as revealed by its efficient cross-linking by psoralen. Two cross-linking sites in the helical region were identified. Mg2+ induced a conformational change of pRNA that exposes the portal protein binding site by promoting the refolding of two strands of the procapsid binding region, resulting in the formation of pRNA-procapsid complexes. The procapsid binding region in this binding-competent conformation could not be cross-linked with psoralen. When the two strands of the procapsid binding region were fastened by cross-linking, pRNA could neither bind procapsids nor package phi29 DNA. A pRNA conformational change was also discernible by comparison of migration rates in native EDTA and Mg2+ polyacrylamide gel electrophoresis and was revealed by T1 RNase probing. The Mg2+ concentration required for the detection of a change in pRNA cross-linking patterns was 1 mM, which was the same as that required for pRNA-procapsid complex formation and DNA packaging and was also close to that in normal host cells.

Bacillus Phages↗

PubMed2006

Chromatin modifications in DNA repair.

A requirement of nuclear processes that use DNA as a substrate is the manipulation of chromatin in which the DNA is packaged. Chromatin modifications cause alterations of histones and DNA, and result in a permissive chromatin environment for these nuclear processes. Recent advances in the fields of DNA repair and chromatin reveal that both histone modifications and chromatin-remodeling complexes are essential for the repair of DNA lesions, such as DNA double strand breaks (DSBs). In particular, chromatin-modifying complexes, such as the INO80, SWR1, RSC, and SWI/SNF ATP-dependent chromatin-remodeling complexes and the NuA4 and Tip60 histone acetyltransferase complexes are implicated in DNA repair. The activity of these chromatin-modifying complexes influences the efficiency of the DNA repair process, which ultimately affects genome integrity and carcinogenesis. Thus, the process of DNA repair requires the cooperative activities of evolutionarily conserved chromatin-modifying complexes that facilitate the dynamic chromatin alterations needed during repair of DNA damage.

Animals↗

PubMed1975

Appearance of transducing particles and the fate of host DNA after infection of Salmonella typhimurium with P22-mutants with increased transducing ability (HT-mutants).

The kinetics of production of transducing particles for different bacterial markers were followed by premature lysis of Salmonella typhimurium cells infected with P22 phages. The were compared for cells infected with wild type phage or with HT-mutants which show increased transduction frequencies. Measuring the sedimentation velocity of bacterial DNA of cells infected with wild type or HT-phages, it was shown that: a) there is no cutting of DNA at random; b) original fragments necessary for packaging host DNA into transducing particles cannot be smaller than 10 phage-genome lengths; c) cutting of transducing fragments leads immediately to the right length; d) there is no loss of precipitable DNA due to phage infection.

DNA, Bacterial↗

PubMed1998

The cenpB gene is not essential in mice.

Centromere protein B (CENP-B) is a centromeric DNA-binding protein that binds to alpha-satellite DNA at the 17 bp CENP-B box sequence. The binding of CENP-B, along with other proteins, to alpha-satellite DNA sequences at the centromere, is thought to package the DNA into heterochromatin subjacent to the kinetochore of mitotic chromosomes. To determine the importance of CENP-B to kinetochore assembly and function, we generated a mouse null for the cenpB gene. The deletion removed part of the promoter and the entire coding sequence except for the carboxyl-terminal 35 amino acids of the CENP-B polypeptide. Mice heterozygous or homozygous for the cenpB null mutation are viable and healthy, with no apparent defect in growth and morphology. We have established mouse embryo fibroblasts from heterozygous and homozygous cenpB null littermates. Microscopic analysis, using immunofluorescence and electron microscopy of the cultured cells, indicated that the centromere-kinetochore complex was intact and identical to control cells. Mitosis was identical in fibroblasts derived from cenpB wild-type, heterozygous and null animals. Our studies demonstrate that CENP-B is not required for the assembly of heterochromatin or the kinetochore, or for completion of mitosis.

Animals↗

PubMed1977