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1.
The structure of a DNA unwinding protein and its complexes with oligodeoxynucleotides by x-ray diffraction. 总被引:1,自引:0,他引:1
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A McPherson F Jurnak A Wang F Kolpak A Rich I Molineux P Fitzgerald 《Biophysical journal》1980,32(1):155-173
The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been solved to 2.3 A resolution by x-ray diffraction techniques. The molecule contains an extensive cleft region that we have identified as the DNA binding site on the basis of the residues that comprise its surface. The interior of the groove has a rather large number of basic amino acid residues that serve to draw the polynucleotide backbone into the cleft. Arrayed along the external edges of the groove are a number of aromatic amino acid side groups that are in position to stack upon the bases of the DNA and fix it in place. The cleft then acts as an elongated pair of jaws that draws the DNA between them by charge interactions involving the phosphates with the interior lysines and arginines. The jaws then close on the DNA strand through small conformation changes and the rotation of aromatic side-chains into position to stack upon the purines and pyrimidines. Complexes of the gene 5 protein with a variety of oligodeoxynucleotides have been formed and crystallized for x-ray diffraction analysis. The crystallographic parameters of four different unit cells indicate that the fundamental unit of the complex is composed of six gene 5 protein dimers. We believe this aggregate has 622 point group symmetry and is a ring formed by end to end closure of a linear array of six dimers. From our results we have proposed a double helical model for the gene 5 protein-DNA complex in which the protein forms a spindle or core around which the DNA is spooled. 5.0-A x-ray diffraction data from one of the crystalline complexes is currently being analyzed by molecular replacement techniques to obtain what we believe will be the first direct visualization of a protein-deoxyribonucleic acid complex approaching atomic resolution. 相似文献
2.
X-ray diffraction studies on crystalline complexes of the gene 5 DNA-unwinding protein with deoxyoligonucleotides 总被引:2,自引:0,他引:2
A McPherson A H Wang F A Jurnak I Molineux F Kolpak A Rich 《The Journal of biological chemistry》1980,255(7):3174-3177
Complexes of the gene 5 protein with a variety of oligodeoxynucleotides, ranging in length from two to eight and having several different sequences, have been formed and crystallized for x-ray diffraction analysis. The crystallographic parameters of four different unit cells, all of which are based on hexagonal packing arrangements, indicate that the fundamental unit of the complex is composed of 12 gene 5 monomers. 相似文献
3.
C. Helene F. Toulme M. Charlier M. Yaniv 《Biochemical and biophysical research communications》1976,71(1):91-98
The binding of denatured DNA to the protein coded by gene 32 of phage T 4 is accompanied by a quenching of the fluorescence of the protein tryptophyl residues. Gene 32 protein also binds to UV-irradiated DNA and photosensitizes the splitting of thymine dimers. Thymine bases are regenerated by this photosensitized reaction both in double stranded and in heat denatured DNA. No photosensitized splitting of thymine dimers is observed when the complex formed by gene 32 protein with UV-irradiated DNA is dissociated at high ionic strength. These results are discussed with respect to the possible stacking interaction of tryptophyl residues of gene 32 protein with bases in single stranded DNA. 相似文献
4.
The beta recombinase of plasmid pSM19035 binds to two adjacent sites, making different contacts at each of them.
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The beta recombinase from plasmid pSM19035 catalyzes intramolecular site-specific recombination between two directly or inversely oriented six sites in the presence of a chromatin-associated protein (Hbsu, HU or HMG-1). The six site is a DNA segment containing two binding sites (I and II) for beta protein dimers. We show that beta recombinase binds sequentially to both sites, having a different affinity for each one. Hydroxyl radical footprints show a different protection pattern at each site. Positions critical for beta protein binding have been identified by methylation interference and missing nucleoside assays. The results indicate that the protein recognizes each site in a different way. Comparison of the beta protein recombination site with that of DNA resolvases and DNA invertases of the Tn3 family, to which it belongs, shows that these sequences can be divided into two regions. One corresponds to the crossover point and is similar for all recombinases of the family. The other region differs in the different subfamilies and seems to have an architectural role in aligning the crossover sites at the synaptic complex. The different ways to assemble this complex could explain why each system leads to a particular recombination event: DNA resolution (resolvases), inversion (invertases) or both (beta recombinase). 相似文献
5.
Nuria A. Illán-Cabeza Rosario A. Vilaplana Ysmael Alvarez Khalid Akdi Sanae Kamah Francisco Hueso-Ureña Miguel Quirós Francisco González-Vílchez Miguel N. Moreno-Carretero 《Journal of biological inorganic chemistry》2005,10(8):924-934
The new complex formed by Cd(II) and the 1:2 Schiff-base-type ligand 2,6-bis[1-(4-amino-1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxopyrimidin-5-yl)imino]ethylpyridine
(DAPDAAU) has been chemically and structurally characterized by X-ray diffraction: the ion Cd(II) is surrounded by six nitrogen
atoms from two DAPDAAU ligands which coordinates each one in a tridentate fashion through the pyridine ring (N1) and both
azomethine nitrogen atoms (N5). The interaction of the Cd(II) complex (compound I) with calf-thymus DNA as observed by circular
dichroism spectroscopy suggests the initial unwinding of the DNA double helix strongly depends on increasing incubation times
and metal-to-nucleic acid molar ratios. Electrophoretic experiments indicate that the cadmium complex induces cleavage of
the plasmid pBR322 DNA to give ulterior nicking and shortening of this molecule, as a result of the complex binding to DNA,
resulting in the conclusion that compound I behaves as a chemical nuclease. Cytotoxic activity of the Cd(II) complex against
selected different human cancer cell lines is specific and increases with increasing concentration of the metal compound;
this fact indicates the potential antitumor character of the complex. When the culture medium is supplemented with compound
I, a remarkable inhibition of the growing cell is observed, important cell degeneration appears before 48 h and abundant precipitates
are formed that correspond to cell residues and denatured proteins.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献
6.
Crystallization and preliminary diffraction data for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii 总被引:6,自引:0,他引:6
Pink crystals of methylmalonyl-CoA mutase from Propionibacterium shermanii, a coenzyme B12 (5'-deoxyadenosylcobalamin)-dependent enzyme, have been obtained by the hanging-drop method in two different forms. One form lies in the space group P21, with unit cell dimensions a = 122 A, b = 160 A and c = 90 A, with beta = 104 degrees (1 A = 0.1 nm). There are two alpha beta dimers in the asymmetric unit. The crystals diffract to 3.2 A resolution and are suitable for high resolution X-ray diffraction studies. 相似文献
7.
The structure of the gene 5 DNA unwinding protein from bacteriophage fd has been determined by X-ray diffraction analysis of single crystals to 2.3 Å resolution using six isomorphous heavy-atom derivatives. The essentially globular monomer appears to consist of three secondary structural elements, a radically twisted three-stranded antiparallel β sheet and two distinct anti-parallel β loops, which are joined by short segments of extended polypeptide chain. The molecule contains no α-helix. A long groove, or arch, 30 Å in length is formed by the underside of the twisted β sheet and one of the two β ribbons. We believe this groove to be the DNA binding region, and this is supported by the assignment of residues on its surface implicated in binding by solution studies. These residues include several aromatic amino acids which may intercalate or stack upon the bases of the DNA. Two monomers are maintained as a dimer by the very close interaction of symmetry related β ribbons about the molecular dyad. About six residues at the amino and carboxyl terminus are in extended conformation and both seem to exhibit some degree of disorder. The amimo-terminal methionine is the locus for binding the platinum heavy-atom derivatives and tyrosine 26 for attachment of the major iodine substituent. 相似文献
8.
Flora S. Groothuizen Alexander Fish Maxim V. Petoukhov Annet Reumer Laura Manelyte Herrie H. K. Winterwerp Martin G. Marinus Joyce H. G. Lebbink Dmitri I. Svergun Peter Friedhoff Titia K. Sixma 《Nucleic acids research》2013,41(17):8166-8181
The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair. 相似文献
9.
Reorganization of terminator DNA upon binding replication terminator protein: implications for the functional replication fork arrest complex.
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Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the replication terminator protein (RTP) and DNA terminator sites. While determination of the crystal structure of RTP has facilitated our understanding of how a single RTP dimer interacts with terminator DNA, additional information is required in order to understand the assembly of a functional fork arrest complex, which requires an interaction between two RTP dimers and the terminator site. In this study, we show that the conformation of the major B.subtilis DNA terminator,TerI, becomes considerably distorted upon binding RTP. Binding of the first dimer of RTP to the B site of TerI causes the DNA to become slightly unwound and bent by approximately 40 degrees. Binding of a second dimer of RTP to the A site causes the bend angle to increase to approximately 60 degrees . We have used this new data to construct two plausible models that might explain how the ternary terminator complex can block DNA replication in a polar manner. In the first model, polarity of action is a consequence of the two RTP-DNA half-sites having different conformations. These different conformations result from different RTP-DNA contacts at each half-site (due to the intrinsic asymmetry of the terminator DNA), as well as interactions (direct or indirect) between the RTP dimers on the DNA. In the second model, polar fork arrest activity is a consequence of the different affinities of RTP for the A and B sites of the terminator DNA, modulated significantly by direct or indirect interactions between the RTP dimers. 相似文献
10.
Complex of bacteriophage M13 single-stranded DNA and gene 5 protein 总被引:19,自引:0,他引:19
Lysates of bacteriophage M13-infected cells contain numerous unbranched filamentous structures approximately 1·1 μm long × 160 Å wide, that is, slightly longer and considerably wider than M13 virions. These structures are complexes of viral single-stranded DNA molecules with M13 gene 5 protein, a non-capsid protein required for single-stranded DNA production. All, or nearly all, of the single-stranded DNA from the infected cells and at least half to two-thirds of the gene 5 protein molecules are found as complex in the lysates. The complex contains about 1300 gene 5 protein molecules per DNA molecule but little if any of the two known capsid proteins. The complex is much less stable than virions in the presence of salt or ionic detergent solutions and in electron micrographs it appears to have a much looser and more open structure. If an excess of M13 single-stranded DNA is added to complex in a lysate, the gene 5 protein molecules from the complex redistribute onto all of the added as well as the original DNA, again suggesting a rather loose association of protein and DNA.By electron microscopy, the complex from infected cells appears to differ structurally from complex formed in vitro between purified single-stranded DNA and purified gene 5 protein. Because of this apparent structural difference and because previous experiments suggested the presence of complex in vivo, we presume that the complex which we have found in lysates of infected cells previously did exist as such inside the cells, but we have been unable to exclude that it formed during or after lysis. If it is assumed that complex does occur in vivo, the results of pulse-chase radioactive labeling experiments on infected cells can be interpreted as showing that with time the single-stranded DNA leaves complex, presumably to be matured into virions, while the gene 5 protein molecules are re-used to form more complex. 相似文献
11.
G D Brayer 《Journal of biomolecular structure & dynamics》1987,4(5):859-868
A modelling procedure has been utilized to obtain a preliminary three-dimensional structural model for the bacteriophage IKe DNA binding protein (IKe-DBP) based on the known high resolution X-ray diffraction structure of a functionally related protein (G5BP) from bacteriophage fd. The degree of structural homology observed is much higher than the 44% primary sequence identity between these proteins would indicate. These studies suggest IKe-DBP, like G5BP, is composed of a central three-stranded beta sheet from which protrude three extended beta loops. Furthermore, the IKe-DBP structural model can easily form, without conformational rearrangements, the compact dimer unit that is the functionally active species of G5BP. Structural comparisons show residues conserved in the primary sequence of both proteins tend to cluster in two regions. The first being essential for the maintenance of dimer association. The second about the two DNA binding channels which cross the face of each dimer. Based upon an earlier characterized G5BP-DNA complex, a model for DNA complexation to IKe-DBP is also presented. 相似文献
12.
When influenza A virus infects host cells, its integral matrix protein M2 forms a proton-selective channel in the viral envelope. Although X-ray crystallography and NMR studies using fragment peptides have suggested that M2 stably forms a tetrameric channel irrespective of pH, the oligomeric states of the full-length protein in the living cells have not yet been assessed directly. In the present study, we utilized recently developed stoichiometric analytical methods based on fluorescence resonance energy transfer using coiled-coil labeling technique and spectral imaging, and we examined the relationship between the oligomeric states of full-length M2 and its channel activities in living cells. In contrast to previous models, M2 formed proton-conducting dimers at neutral pH and these dimers were converted to tetramers at acidic pH. The antiviral drug amantadine hydrochloride inhibited both tetramerization and channel activity. The removal of cholesterol resulted in a significant decrease in the activity of the dimer. These results indicate that the minimum functional unit of the M2 protein is a dimer, which forms a complex with cholesterol for its function. 相似文献
13.
Susan J. Cavalieri Kenneth E. Neet David A. Goldthwait 《Journal of molecular biology》1976,102(4):697-711
Isolated gene 5 protein from bacteriophage fd-infected Escherichia coli has been shown by sedimentation equilibrium to exist primarily as a dimer under non-denaturing conditions. The dimer was stable under conditions of high ionic strength, extremes in pH, dilution to 0.075 mg/ml, and increased temperature. Gene 5 protein did not undergo the indefinite self-association observed with gene 32 protein.Three lines of evidence for co-operative binding of gene 5 protein to DNA were developed. First, the interaction between gene 5 protein and phage T4 DNA was examined using a nitrocellulose filter assay. Scatchard plots of the binding data indicated that the interaction was co-operative. Similar results were obtained with gene 32 protein. Second, the co-operative binding of both proteins to DNA was shown by the sensitivity of the protein-DNA interaction to increasing ionic strength at various ratios of protein to DNA. Finally, by using the cross-linking agent, dimethyl suberixmidate, oligomeric structures containing at least seven monomers were found when the DNA was less than saturated.The possibility that gene 5 protein dimers undergo indefinite self-association in the presence of oligonucleotides was examined by sedimentation equilibrium. With oligo[d(pT)4], the protein dimer was complexed with this oligonucleotide but no self-association was observed. With oligo[d(pT)8], gene 5 protein formed tetramers, but no significant indefinite association was noted. These results do not suggest a DNA-induced conformational change, which results in indefinite association. A model for the co-operative binding of gene 5 protein to DNA is presented. 相似文献
14.
Crystallization of Escherichia coli catabolite gene activator protein with its DNA binding site. The use of modular DNA 总被引:1,自引:0,他引:1
To obtain crystals of the Escherichia coli catabolite gene activator protein (CAP) complexed with its DNA-binding site, we have searched for crystallization conditions with 26 different DNA segments greater than or equal to 28 base-pairs in length that explore a variety of nucleotide sequences, lengths, and extended 5' or 3' termini. In addition to utilizing uninterrupted asymmetric lac site sequences, we devised a novel approach of synthesizing half-sites that allowed us to efficiently generate symmetric DNA segments with a wide variety of extended termini and lengths in the large size range (greater than or equal to 28 bp) required by this protein. We report three crystal forms that are suitable for X-ray analysis, one of which (crystal form III) gives measurable diffraction amplitudes to 3 A resolution. Additives such as calcium, n-octyl-beta-D-glucopyranoside and spermine produce modest improvements in the quality of diffraction from crystal form III. Adequate stabilization of crystal form III is unexpectedly complex, requiring a greater than tenfold reduction in the salt concentration followed by addition of 2-methyl-2,4-pentanediol and then an increase in the concentration of polyethylene glycol. 相似文献
15.
16.
17.
Sequence and hairpin structure of an inverted repeat series at termini of the Physarum extrachromosomal rDNA molecule 总被引:6,自引:0,他引:6
The termini of the 61 kb palindromic rDNA molecules of Physarum polycephalum possess a series of multiple inverted repeats in which are located specific single-strand gaps and tightly attached protein. After treating rDNA with S1 nuclease, we have cloned several 5 kb Eco RI terminal restriction fragments. Sequencing of more than 800 nucleotides from the end of one such clone reveals the presence of six to ten tandemly repeated units averaging 140 +/- 4 bp in length and flanked by Hae III sites. Each 140 nucleotide repeat unit can form thermodynamically stable hairpin structures based on complex internal palindromic components. When the specific gap sequence CCCTA is present, it is located near the apex of a hairpin component. These secondary structures are formed in growing plasmodia, as seen in electron micrographs of native rDNA molecules, which also reveal apparent recombination forms involving rDNA ends and noncontiguous DNA segments. Recombination initiated at terminal single-strand hairpin loops can result in genetic exchange of ribosomal gene sequences and can lead to completion of 5' nucleotide sequences at ends of newly replicated rDNA molecules. 相似文献
18.
X-ray fibre diffraction and scanning transmission electron microscopy have been used to investigate the structure of an intracellular complex between circular single-stranded viral DNA and a viral DNA-binding protein. This complex is an intermediate between replication and assembly of the filamentous bacteriophage Pf1. By scanning transmission electron microscopy, the complex has a length of 1.00 μm and Mr = 29.6 × 106. It consists of 1770 protein subunits, each of 15,400 Mr, and one viral DNA molecule of 2.3 × 106Mr: there are 4.2 ± 0.5 nucleotides per subunit. The structure is flexible in solution, but in oriented dry fibres it forms a regular helix of 45 Å pitch having 6.0 dimeric protein subunits per turn, with an axial spacing of 7.5 Å between dimers and 1.9 Å between adjacent nucleotides. Model calculations suggest that the protein dimers may be oriented in a direction approximately perpendicular to the 45 Å helix, so that each dimer spans the two anti-parallel DNA chains. The results imply that conformational changes are required of the DNA as it is transferred from the double-stranded form to the replication-assembly complex, and subsequently to the virion. 相似文献
19.
Gene 4 of bacteriophage T7 encodes two proteins, a 63-kDa protein and a colinear 56-kDa protein, that are essential for synthesis of leading and lagging strands during DNA replication. The gene 4 proteins together catalyze the synthesis of oligoribonucleotides, pppACC(C/A) or pppACAC, at the single-stranded DNA sequences 3'-CTGG(G/T)-5' or 3'-CTGTG-5', respectively. Purified 56-kDa protein has helicase activity, but no primase activity. In order to study 63-kDa gene 4 protein free of 56-kDa gene 4 protein, mutations were introduced into the internal ribosome-binding site responsible for the translation of the 56-kDa protein. The 63-kDa gene 4 protein was purified 16,000-fold from Escherichia coli cells harboring an expression vector containing the mutated gene 4. Purified 63-kDa gene 4 protein has primase, helicase, and single-stranded DNA-dependent dTTPase activities. The constraints of primase recognition sequences, nucleotide substrate requirements, and the effects of additional proteins on oligoribonucleotide synthesis by the 63-kDa gene 4 protein have been examined using templates of defined sequence. A three-base sequence, 3'-CTG-5', is necessary and sufficient to support the synthesis of pppAC dimers. dTTP hydrolysis is essential for oligoribonucleotide synthesis. Addition of a 7-fold molar excess of 56-kDa gene 4 protein to 63-kDa protein increases the number of oligoribonucleotides synthesized by 63-kDa protein 100-fold. The increase in oligonucleotides results predominantly from an increase in the synthesis of tetramers, with relatively little change in the synthesis of dimers and trimers. The presence of 56-kDa protein also causes 63-kDa protein to synthesize "pseudo-templated" pppACCCC pentamers at the recognition sequence 3'-CTGGG-5'. T7 gene 2.5 protein, a single-stranded DNA binding protein, increases the total number of oligoribonucleotides synthesized by 63-kDa gene 4 protein on single-stranded M13 DNA, but has no effect on the ratio of dimers to trimers and tetramers. 相似文献
20.
Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 总被引:3,自引:0,他引:3
The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers. 相似文献