Adenovirus terminal protein protects single stranded DNA from digestion by a cellular exonuclease.

Adenovirus 5 DNA-protein complex is isolated from virions as a duplex DNA molecule covalently attached by the 5' termini of each strand to virion protein of unknown function. The DNA-protein complex can be digested with E. coli exonuclease III to generate molecules analogous to DNA replication intermediates in that they contain long single stranded regions ending in 5' termini bound to terminal protein. The infectivity of pronase digested Adenovirus 5 DNA is greatly diminished by exonuclease III digestion. However, the infectivity of the DNA-protein complex is not significantly altered when up to at least 2400 nucleotides are removed from the 3' ends of each strand. This indicates that the terminal protein protects 5' terminated single stranded regions from digestion by a cellular exonuclease. DNA-protein complex prepared from a host range mutant with a mutation mapping in the left 4% of the genome was digested with exonuclease III, hybridized to a wild type restriction fragment comprising the left 8% of the genome, and transfected into HeLa cells. Virus with wild type phenotype was recovered at high frequency.     

Characterization of a protein covalently linked to the 5' termini of the DNA of Bacillus subtilis phage phi29.

We have isolated a covalent DNA-protein complex from bacteriophage φ29 particles. Polyacrylamide gel electrophoresis and tryptic peptide analysis showed that the protein present in the complex is very similar or identical to p3, an early induced protein essential for viral DNA replication.When the DNA-protein complex is treated with the restriction endonuclease EcoRI, the protein is specifically associated to the two terminal fragments, A and C. The protein is probably linked to the 5′ termini of the DNA since proteinase K-treated DNA is resistant to phosphorylation with polynucleotide kinase, even after treatment with alkaline phosphatase, while it is sensitive to exonuclease III. By electron microscopy the protein is visualized as a dot located at the ends of unit length DNA molecules.Mixed infection of Bacillus subtilis, at 42 °C, with ts² mutants in cistrons 2 and 3 only produces ts 2 progeny. This finding suggests that an inactive protein p3 bound to the DNA of the ts 3 mutant is not replaced by a functional protein and, as a consequence, replication of the ts 3 DNA does not occur.     

CAP and RNA polymerase interactions with the lac promoter: binding stoichiometry and long range effects.

The binding stoichiometries of the complexes formed when the E. coli cyclic AMP receptor protein (CAP) binds to 203 bp lac promoter-operator restriction fragments have been determined. Under quantitative binding conditions, a single dimer of CAP occupies each of two sites in the promoter. Different electrophoretic mobilities are observed for 1:1 complexes formed with L8-UV5 mutant, L305 mutant, and wild type promoter fragments, indicating sequence-specific structural differences between the complexes. The differences in gel mobility between L8-UV5 and wild type complexes disappear when the promoter fragments are cleaved with Hpa II restriction endonuclease. Models in which CAP alters DNA conformation or in which CAP forms a transient intramolecular bridge between two domains of a DNA molecule could account for these observations. The selective binding of RNA polymerase to CAP-promoter complexes is demonstrated: the binding of a single CAP dimer to the promoter is sufficient to stimulate subsequent polymerase binding. Functional CAP molecules are not released from the promoter on polymerase binding.     

Identification of a protein linked to the ends of adenovirus DNA.

A DNA-protein complex from human adenoviruses has been further characterized by electron microscopy, radiochemical labeling and analytical ultracentrifugation. Preparations of the complex contain a large percentage of forms of DNA which are either circular or oligomeric and are readily distinguishable from preparations of pronase-treated adenovirus DNA by analytical ultracentrifugation in cesium chloride gradients containing 4 M guanidinium chloride. The protein component has been iodinated in vitro with ¹²⁵I using Bolton and Hunter reagent, and SDS-polyacrylamide gel analysis of the labeled protein indicates that it has an apparent molecular weight of 55,000 daltons. DNAase I digestion of the DNA-protein complex labeled with ³²PO₄ results in release of a ³²PO₄-labeled protein which remains labeled even after boiling in 1% SDS and 1% mercaptoethanol. Subsequent digestion of this entity with snake venom phosphodiesterase leads to release of ³²P₄-labeled 5′-phosphate deoxynucleotides. Digestion of the DNA-protein complex with Eco R1 and analysis of the isolated restriction fragments indicates that the protein is present on each terminal fragment. We conclude that there is a protein of 55,000 daltons directly attached to each 5′ end of molecule probably via a covalent linkage. We propose that the protein functions during DNA replication by facilitating priming of the progeny strands, thus allowing the 5′ ends of the DNA to be replicated.     

Crystal structure of a cyclic AMP-independent mutant of catabolite gene activator protein

Escherichia coli NCR91 synthesizes a mutant form of catabolite gene activator protein (CAP) in which alanine 144 is replaced by threonine. This mutant, which also lacks adenylate cyclase activity, has a CAP phenotype; in the absence of cAMP it is able to express genes that normally require cAMP. CAP91 has been purified and crystallized with cAMP under the same conditions as used to crystallize the wild type CAP X cAMP complex. X-ray diffraction data were measured to 2.4-A resolution and the CAP91 structure was determined using initial model phases from the wild type structure. A difference Fourier map calculated between CAP91 and wild type showed the 2 alanine to threonine sequence changes in the dimer and also a change in orientation of cysteine 178 in one of the subunits. The CAP91 coordinates were refined by restrained least squares to an R factor of 0.186. Differences in the atomic positions of the wild type and mutant protein structures were analyzed by a vector averaging technique. There were small changes that included concerted motions in the small domains, in the hinge between the two domains and in an adjacent loop between beta-strands 4 and 5. The mutation at residue 144 apparently causes changes in the position of some protein atoms that are distal to the mutation site.     

Open strands adjacent to iterons promote the binding of the replication initiator protein (Rep) of pSC101 to the unit sequence of the iterons in vitro

The purified dimeric form of the Rep protein, a replication initiator protein of the plasmid pSC101, has a low affinity for repeated sequences, iterons, in the replication origin of the plasmid, and higher affinities for two inverted repeats in the operator region of the rep gene resulting in its functioning as an autorepressor. Studies of binding to various synthetic DNA have established that Rep can bind to duplex iteron-sequence carrying open (non-complementary) strands at one end proximal to the rep gene. Open strands at the opposite end of the iteron have no effect on Rep-binding. One open strand seems to be required in a sequence-specific fashion. A randomly sequenced duplex DNA with the open strands cannot bind to Rep but can function as a significant competitor. This suggests that Rep has some affinity for the open strands and forms a stable complex with the adjacent iteron. The mutated Rep protein, Rep1, which causes an increase in the plasmid copy number in vivo, has equally high affinity for the iteron with the open strands as wild type Rep, though it has a lower affinity for the inverted repeats than the wild type. The Rep dimer might bind to these DNA sequences with different modes.     

Identification of tyrosine 204 as the photo-cross-linking site in the DNA-EcoRI DNA methyltransferase complex by electrospray ionization mass spectrometry

We describe a highly sensitive strategy combining laser-induced photo-cross-linking and HPLC-based electrospray ionization mass spectrometry to identify amino acid residues involved in protein-DNA recognition. The photoactivatible cross-linking thymine isostere, 5-iodoracil, was incorporated at a single site within the sequence recognized by EcoRI DNA methyltransferase (GAATTC). UV irradiation of the DNA-protein complex at 313 nm results in a >60% cross-linking yield. SDS-polyacrylamide gel electrophoresis and mass spectrometry were used to analyze the covalent cross-linked complex. The total mass is consistent with covalent bond formation between one strand of DNA and the protein with 1:1 stoichiometry. Protease digestion of the cross-linked complex yields several peptide-DNA adducts that were purified by anion-exchange column chromatography. A combination of mass spectrometric analysis and amino acid sequencing revealed that tyrosine 204 was cross-linked to the DNA. Electrospray mass spectrometric analysis of the peptide-nucleoside adduct confirmed this assignment. Tyrosine 204 resides in a peptide motif previously thought to be involved in AdoMet binding and methyl transfer. Thus, amino acids within loop segments but outside of "DNA binding" motifs can be critical to DNA recognition. Our method provides an accurate characterization of picomole quantities of DNA-protein complexes.     

Effects of 4-nitroquinoline 1-oxide on the DNA-protein complex

The effects of 4-nitroquinoline 1-oxide (4NQO), a well known carcinogenic compound, on the DNA-protein complex and DNA Folding Proteins were investigated. FM3A cells were treated with 10(-6) M or 10(-5) M 4NQO for 30 min. Treatment with the 10(-6) M concentration was confirmed to cause the sedimentation of the DNA-protein complex to become slower. DNA Folding Proteins were then isolated from 4NQO-treated and untreated control cells and analyzed by SDS-polyacrylamide gel electrophoresis. No appreciable differences in the amounts of the major components of DNA Folding Proteins could be found due to 4NQO-treatment, but the 92 K protein was induced in DNA-protein complex by treatment with 4NQO.     

Fluorescence microscopy of DNA-protein structures from osmotically lysed mitochondria of yeast and tobacco

Summary The mitochondrial nucleoid is a compact structure composed of DNA and protein. By fluorescence microscopy, decondensation of the nucleoids was observed when yeast and tobacco mitochondria were osmotically lysed and subjected to an electric field. Structures stained with ethidium bromide were seen moving toward either the anode or the cathode. Since the movement of deproteinized DNA is toward the anode, the structures moving toward the cathode represent DNA-protein complexes with a net positive charge. Nucleoid decondensation and unfolding of the DNA probably resulted from the removal of weakly bound proteins; yet high-affinity basic proteins were evidently retained yielding cationic DNA-protein structures. Some of the positively charged structures were observed to break, presumably at single-stranded DNA regions, releasing negatively charged particles. The DNA-protein structures were complex branching forms larger than the unit genome, suggesting that multigenomic, concatemeric DNA is present within the mitochondria.Abbreviations DAPI 4,6-diamidino-2-phenylindole - EtBr ethidium bromide - HMG high-mobility group - mt-genome mitochondrial genome - mt-nucleoid mitochondrial nucleoid - PFGE pulsed-field gel electrophoresis - pt-nucleoid plastid nucleoid - ssDNA single-stranded DNA     

Genome-linked protein associated with the 5' termini of bacteriophage phi29 DNA.

A DNA-protein complex was isolated from Bacillus subtilis bacteriophage phi29 by sucrose gradient sedimentation or gel filtration in the presence of agents known to break noncovalent bonds. A 28,000-dalton protein was released from this complex by subsequent hydrolysis of the DNA. The DNA-protein complex was examined for its susceptibility to enzymes which act upon the 5' and 3' termini of DNA molecules. It was susceptible to exonucleolytic degradation from the 3' termini by exonuclease III but not from the 5' termini by lambda exonuclease. Attempts to label radioactively the 5' termini by phosphorylation with T4 polynucleotide kinase were unsuccessful despite prior treatment with alkaline phosphatase or phosphatase treatment of denatured DNA. Removal of the majority of the bound protein by proteolytic digestion did not increase susceptibility. These results suggest that the linked protein is covalently attached to the 5' termini of phi29 DNA.     

The 1.6Å resolution structure of activated D138L mutant of catabolite gene activator protein with two cAMP bound in each monomer

The X-ray crystal structure of the cAMP-liganded D138L mutant of Escherichia coli catabolite gene activator protein (CAP) was determined at a resolution of 1.66?. This high resolution crystal structure reveals four cAMP binding sites in the homodimer. Two anti conformations of cAMPs (anti-cAMP) locate between the β-barrel and the C-helix of each subunit; two syn conformations of cAMPs (syn-cAMP) bind on the surface of the C-terminal domain. With two syn-cAMP molecules bound, the D138L CAP is highly symmetrical with both subunits assuming a "closed" conformation. These differences make the hinge region of the mutant more flexible. Protease susceptibility measurements indicate that D138L is more susceptible to proteases than that of wild type (WT) CAP. The results of protein dynamic experiments (H/D exchange measurements) indicate that the structure of D138L mutant is more dynamic than that of WT CAP, which may impact the recognition of specific DNA sequences.     

Cation binding linked to a sequence-specific CAP-DNA interaction

The equilibrium association constant observed for many DNA-protein interactions in vitro (K(obs)) is strongly dependent on the salt concentration of the reaction buffer ([MX]). This dependence is often used to estimate the number of ionic contacts between protein and DNA by assuming that release of cations from the DNA is the dominant involvement of ions in the binding reaction. With this assumption, the graph of logK(obs) versus log[MX] is predicted to have a constant slope proportional to the number of ions released from the DNA upon protein binding. However, experimental data often deviate from log-linearity at low salt concentrations. Here we show that for the sequence-specific interaction of CAP with its primary site in the lactose promoter, ionic stoichiometries depend strongly on cation identity and weakly on anion identity. This outcome is consistent with a simple linkage model in which cation binding by the protein accompanies its association with DNA. The order of ion affinities deduced from analysis of DNA binding is the same as that inferred from urea-denaturation experiments performed in the absence of DNA, suggesting that ion binding to free CAP contributes significantly to the ionic stoichiometry of DNA binding. In living cells, the coupling of ion-uptake and DNA binding mechanisms could reduce the sensitivity of gene-regulatory interactions to changes in environmental salt concentration.     

Specific association of a small protein with the telomeric DNA-protein complex during the onset of leaf senescence in Arabidopsis thaliana

Telomeres and their changes in length throughout the life span of cells have been intensively investigated in different organisms. Telomere length is assumed to control replicative senescence in mammalian cells. However, only very few data are available on the developmental dynamics of plant telomeres. Here, changes of telomere length and DNA-protein structure of Arabidopsis thaliana telomeres were analysed in different stages of development, with the main focus resting on the transition from pre-senescent to senescent leaves. The lengths of the telomeres, ranging from ca. 2.0 to 6.5 kb, do not significantly change during plant development indicating that telomere length is not involved in differentiation and replicative senescence nor in post-mitotic senescence of A. thaliana. In dedifferentiated cultured cells a slight increase in length can be determined. The nucleoprotein structure of the telomeric DNA was investigated by gel mobility shift assays, with synthetic oligonucleotides and nuclear protein extracts derived from four defined stages of post-mitotic leaf senescence. In all four stages, a highly salt-resistant DNA-protein complex was formed with the double-stranded as well as with the single-stranded G-rich telomeric DNA. An additional DNA-protein complex was identified in nuclear protein extracts isolated from plants in the transition stage from pre-senescence to senescence. The protein components of the DNA-protein complexes were analysed on native PAGE and SDS-PAGE gels. A protein of 67 kDa (ATBP1) bound to the telomeric DNA in all developmental stages. An additional protein of merely 22 kDa (ATBP2) was associated via protein-protein interaction with ATBP1 to form a higher-order complex exclusively during the onset of senescence. DNA interaction of this higher-order protein complex seems to be restricted to double-stranded telomeric DNA. The defined period of ATBP1/ATBP2 complex formation with the telomeric DNA probably indicates that ATBP2 is involved in the onset of post-mitotic leaf senescence by either disturbing an established or establishing an additional function exhibited by the telomeres in the interphase nuclei.     

A protein fraction stably linked to DNA in plant chromatin

DNA from the chromatin of roots and shoots of maize seedlings was isolated and extensively deproteinized by repeated high-salt extractions, by subsequent deproteinizations eliminating noncovalently associated proteins and by CsC1 density gradient centrifugation. Nevertheless, a protein component resisting all extraction procedures was found firmly associated to plant nuclear DNA. This component was responsible for the ¹²⁵I uptake when a DNA preparation had been labeled by the chloramine-T method.A residual oligodeoxynucleotide-oligopeptide complex was obtained after extensive digestions of the initial DNA-protein complex with proteases and nucleases. The stability of this complex to different chemical treatments suggested a phosphoester type of a linkage. The hydrolysis of this complex by phosphodiesterases indicated that the protein component was linked to plant chromosomal DNA through a phosphodiester bond formed by a hydroxyaminoacid and a 5-end DNA phosphate. Two-dimensional tryptic peptide mapping of the proteins isolated from the two maize chromatins revealed a high degree of similarity to the corresponding proteins of animal origin. Its conservative structure suggests an important role for this protein component in the functioning of the eukaryotic genome.     

Enhanced infectivity of adenovirus type 2 DNA and a DNA-protein complex.

The infectivity of adenovirus type 2 DNA and a DNA-protein complex was studied in 293 cells, a human embryonic kidney cell line transformed by sheared adenovirus type 5 DNA, and in human KB cells. Adenovirus type 2 DNA was more infectious (up to about 40-fold) in 293 cells than in KB cells, whereas a DNA-protein complex (prepared by a rapid procedure) had about the same infectivity in both cell lines. These data may mean that a factor present in 293 cells (perhaps a viral-coded protein) enhances the infectivity of free viral DNA. The infectivity of DNA and the DNA-protein complex was increased up to fivefold by brief treatment of cell monolayers with 25% dimethyl sulfoxide after transfection. Under these conditions, (i) the infectivity of native adenovirus type 2 DNA ranged from 400 to 1,300 PFU/microgram of DNA in 293 cells and from about 9 to 14 PFU/microgram of DNA in KB cells, and (ii) the infectivity of the DNA-protein complex was 6 X 10(3)to 2 X 10(4) PFU/microgram in 293 cells and 1.4 X 10(4) to 1.6 X 10(4) PFU/microgram in KB cells.