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1.
Structure of helical RecA-DNA complexes. II. Local conformational changes visualized in bundles of RecA-ATP gamma S filaments 总被引:4,自引:0,他引:4
Complexes of RecA-DNA filaments, formed in the presence of a non-hydrolyzable ATP analog, ATP gamma S, aggregate together into regular bundles in the presence of Mg2+. Electron micrographs of several different forms of RecA-double-stranded DNA bundles have been analyzed: bundles of six supercoiled filaments at two different concentrations of Mg2+, and bundles of three supercoiled filaments at a single concentration of Mg2+. The bundles are all characterized by a regular left-handed supercoiling of the component filaments arising from the non-integral number of RecA subunits per turn of the RecA helix in these aggregates, about 6.15 units/turn. When single-stranded DNA is used instead of double-stranded DNA, regular aggregates composed of many filaments are formed. These aggregates do not supercoil, consistent with a symmetry of the component filaments of close to 6.0 units/turn. These different structures have provided a strong confirmation of the analysis of isolated RecA filaments. Since different RecA protomers within the component filaments of these aggregates are in different environments, they have provided a direct view of different conformations that RecA subunits may adopt within the same filament as a result of nonequivalent contacts. The conformational changes we have visualized are quite large, with apparent movements of mass over distances greater than 2 nm. The RecA-mediated strand exchange reaction is a highly dynamic process, which involves both the unwinding and stretching of DNA, in addition to the physical movement of DNA strands. It is quite likely, therefore, that the different conformations of RecA subunits seen in these aggregates represent different states of RecA during its enzymatic strand exchange activity. 相似文献
2.
Overproduction of single-stranded-DNA-binding protein specifically inhibits recombination of UV-irradiated bacteriophage DNA in Escherichia coli. 总被引:4,自引:2,他引:2
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P L Moreau 《Journal of bacteriology》1988,170(6):2493-2500
Overproduction of single-stranded DNA (ssDNA)-binding protein (SSB) in uvr Escherichia coli mutants results in a wide range of altered phenotypes. (i) Cell survival after UV irradiation is decreased; (ii) expression of the recA-lexA regulon is slightly reduced after UV irradiation, whereas it is increased without irradiation; and (iii) recombination of UV-damaged lambda DNA is inhibited, whereas recombination of nonirradiated DNA is unaffected. These results are consistent with the idea that in UV-damaged bacteria, SSB is first required to allow the formation of short complexes of RecA protein and ssDNA that mediate cleavage of the LexA protein. However, in a second stage, SSB should be displaced from ssDNA to permit the production of longer RecA-ssDNA nucleoprotein filaments that are required for strand pairing and, hence, recombinational repair. Since bacteria overproducing SSB appear identical in physiological respects to recF mutant bacteria, it is suggested that the RecF protein (alone or with other proteins of the RecF pathway) may help RecA protein to release SSB from ssDNA. 相似文献
3.
In the presence of a molar excess of eukaryotic DNA topoisomerase II and an appropriate concentration of dextran sulfate, relaxed closed circular DNA is converted to a negatively supercoiled form. The reaction is dependent on ATP. Neither adenosine 5'-[beta,gamma-imido]-triphosphate nor adenosine 5'-[gamma-thio]triphosphate can substitute for ATP. The negative supercoils formed are relaxed by subsequent addition of DNA topoisomerase I to the supercoiling reaction mixture. Covalent closure of a nicked circular DNA in the presence of DNA topoisomerase II and dextran sulfate but in the absence of ATP causes a small decrease in the linking number. These results suggest that when an appropriate concentration of dextran sulfate is present, the binding of a molar excess of eukaryotic DNA topoisomerase II constrains a small number of negative supercoils in DNA, which in turn generate unconstrained negative supercoils at the expense of ATP. 相似文献
4.
High positive supercoiling in vitro catalyzed by an ATP and polyethylene glycol-stimulated topoisomerase from Sulfolobus acidocaldarius 总被引:18,自引:1,他引:17
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A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 - 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine-5' -0-(3-thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP-driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.= 相似文献
5.
RecA-mediated annealing of single-stranded DNA and its relation to the mechanism of homologous recombination 总被引:1,自引:0,他引:1
We demonstrate that RecA protein can mediate annealing of complementary DNA strands in vitro by at least two different mechanisms. The first annealing mechanism predominates under conditions where RecA protein causes coaggregation of single-stranded DNA (ssDNA) molecules and where RecA-free ssDNA stretches are present on both reaction partners. Under these conditions annealing can take place between locally concentrated protein-free complementary sequences. Other DNA aggregating agents like histone H1 or ethanol stimulate annealing by the same mechanism. The second mechanism of RecA-mediated annealing of complementary DNA strands is best manifested when preformed saturated RecA-ssDNA complexes interact with protein-free ssDNA. In this case, annealing can occur between the ssDNA strand resident in the complex and the ssDNA strand that interacts with the preformed RecA-ssDNA complex. Here, the action of RecA protein reflects its specific recombination promoting mechanism. This mechanism enables DNA molecules resident in the presynaptic RecA-DNA complexes to be exposed for hydrogen bond formation with DNA molecules contacting the presynaptic RecA-DNA filament. 相似文献
6.
RecA-catalyzed DNA recombination is initiated by a mandatory, high-energy form of DNA in RecA-nucleoprotein filaments, where bases are highly unstacked and the backbone is highly unwound. Interestingly, only the energetics consequent to adenosine triphosphate (ATP) binding, rather than its hydrolysis, seems sufficient to mediate such a high-energy structural hallmark of a recombination filament. The structural consequence of ATP hydrolysis on the DNA part of the filament thus remains largely unknown. We report time-resolved fluorescence dynamics of bases in RecA-DNA complexes and demonstrate that DNA bases in the same exhibit novel, motional dynamics with a rotational correlation time of 7-10 ns, specifically in the presence of ATP hydrolysis. When the ongoing ATP hydrolysis of RecA-DNA filament is "poisoned" by a nonhydrolyzable form of ATP (ATPgammaS), the motional dynamics cease and reveal a global motion with a rotational correlation time of >20 ns. Such ATP hydrolysis-induced flexibility ensues in single-stranded as well as double-stranded bases of RecA-DNA filaments. These results suggest that the role of ATP hydrolysis is to induce a high level of backbone flexibility in RecA-DNA filament, a dynamic property that is likely to be important for efficient strand exchanges in ATP hydrolysis specific RecA reactions. It is the absence of these motions that may cause high rigidity in RecA-DNA filaments in ATPgammaS. Dynamic light scattering measurement comparisons of RecA-ss-DNA filaments formed in ATPgammaS vs that of ATP confirmed such an interpretation, where the former showed a complex of larger (30 nm) hydrodynamic radius than that of latter (12-15 nm). Taken together, these results reveal a more dynamic state of DNA in RecA-DNA filament that is hydrolyzing ATP, which encourage us to model the role of ATP hydrolysis in RecA-mediated DNA transactions. 相似文献
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Control of bacterial DNA supercoiling 总被引:29,自引:0,他引:29
Karl Drlica 《Molecular microbiology》1992,6(4):425-433
Two DNA topoisomerases control the level of negative supercoiling in bacterial cells. DNA gyrase introduces supercoils, and DNA topoisomerase I prevents supercoiling from reaching unacceptably high levels. Perturbations of supercoiling are corrected by the substrate preferences of these topoisomerases with respect to DNA topology and by changes in expression of the genes encoding the enzymes. However, supercoiling changes when the growth environment is altered in ways that also affect cellular energetics. The ratio of [ATP] to [ADP], to which gyrase is sensitive, may be involved in the response of supercoiling to growth conditions. Inside cells, supercoiling is partitioned into two components, superhelical tension and restrained supercoils. Shifts in superhelical tension elicited by nicking or by salt shock do not rapidly change the level of restrained supercoiling. However, a steady-state change in supercoiling caused by mutation of topA does alter both tension and restrained supercoils. This communication between the two compartments may play a role in the control of supercoiling. 相似文献
9.
An assay for visualization of DNA loops undergoing supercoiling changes has been developed. The assay utilizes the fluorescent dye, propidium iodide (PI), which intercalates into the DNA and under the proper conditions causes the supercoiling status of the DNA to change. Thus, the DNA can be seen as a fluorescent halo that changes diameter with PI concentration. At low PI concentrations (0-7.5 micrograms/ml) the supercoils are relaxed with increasing PI, while at higher PI concentrations (7.50-50 micrograms/ml) supercoils in the opposite winding sense are rewound with increasing PI. When HeLa cells were irradiated with 1-20 Gy of 137Cs gamma-rays, the ability to rewind the DNA supercoils was inhibited in a dose-dependent manner, presumably because of the presence of radiation-induced DNA strand breakage, which removed the topological constraints on the DNA loops. These lesions were repaired rapidly during post-irradiation incubation. The ability of the DNA loops to be rewound was restored within 8 min after 10 Gy of gamma-irradiation, such that no difference from control cells could be detected. The half-time for repair of the radiation-induced lesions that inhibit DNA rewinding was similar to that for repair of DNA single strand breaks. The assay has certain advantages over current methods for assaying DNA damage in that it involves measurement of single cells and it does not require the DNA to be labeled with radioactive precursors. 相似文献
10.
Type I restriction enzymes cleave DNA at non-specific sites far from their recognition sequence as a consequence of ATP-dependent DNA translocation past the enzyme. During this reaction, the enzyme remains bound to the recognition sequence and translocates DNA towards itself simultaneously from both directions, generating DNA loops, which appear to be supercoiled when visualised by electron microscopy. To further investigate the mechanism of DNA translocation by type I restriction enzymes, we have probed the reaction intermediates with DNA topoisomerases. A DNA cleavage-deficient mutant of EcoAI, which has normal DNA translocation and ATPase activities, was used in these DNA supercoiling assays. In the presence of eubacterial DNA topoisomerase I, which specifically removes negative supercoils, the EcoAI mutant introduced positive supercoils into relaxed plasmid DNA substrate in a reaction dependent on ATP hydrolysis. The same DNA supercoiling activity followed by DNA cleavage was observed with the wild-type EcoAI endonuclease. Positive supercoils were not seen when eubacterial DNA topoisomerase I was replaced by eukaryotic DNA topoisomerase I, which removes both positive and negative supercoils. Furthermore, addition of eukaryotic DNA topoisomerase I to the product of the supercoiling reaction resulted in its rapid relaxation. These results are consistent with a model in which EcoAI translocation along the helical path of closed circular DNA duplex simultaneously generates positive supercoils ahead and negative supercoils behind the moving complex in the contracting and expanding DNA loops, respectively. In addition, we show that the highly positively supercoiled DNA generated by the EcoAI mutant is cleaved by EcoAI wild-type endonuclease much more slowly than relaxed DNA. This suggests that the topological changes in the DNA substrate associated with DNA translocation by type I restriction enzymes do not appear to be the trigger for DNA cleavage. 相似文献
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12.
Visualization of recA protein and its association with DNA: a priming effect of single-strand-binding protein 总被引:14,自引:0,他引:14
A stoichiometric interaction of RecA protein with single-stranded DNA promotes homologous pairing of the single strand with duplex DNA and subsequent polar formation of a heteroduplex joint. Escherichia coli single-strand-binding (SSB) protein augments these reactions. Electron microscopic observations suggest structural bases for these interactions. Without triphosphates or DNA, RecA protein forms short linear filaments. With added circular single-stranded DNA, it forms extended circular filaments as well as collapsed and aggregated complexes of protein and DNA. The extended circular filaments are stiff and regular in appearance, contrasting with the convoluted structure formed by SSB protein and single-stranded DNA. Together, these two proteins form mixed filaments, which mostly resemble the extended structures containing RecA protein; moreover, SSB protein accelerates formation of extended filaments more than 50-fold, increasing the yield of these structures at the expense of heterogeneous aggregates. Other observations further define the interactions of RecA protein with partially single-stranded DNA, and the effects of ATP gamma S on the tendency of RecA protein to form polymeric structures even in the absence of DNA. 相似文献
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Reverse gyrase is a unique type IA topoisomerase that can introduce positive supercoils into DNA. We have investigated some of the biochemical properties of Archaeoglobus fulgidus reverse gyrase. It can mediate three distinct supercoiling reactions depending on the adenine nucleotide cofactor that is present in the reaction. Besides the ATP-driven positive supercoiling reaction, the enzyme can introduce negative supercoils with a nonhydrolyzable analog, adenylyl imidodiphosphate. In the presence of ADP the plasmid DNA is relaxed almost completely, leaving a very low level of positive supercoiling. Surprisingly, the final supercoiling extent for all three distinct reactions depends on the stoichiometry of enzyme to DNA. This dependence is not due to the difference of reaction rate, suggesting that the amount of enzyme bound to DNA is an important determinant for the final supercoiling state of the reaction product. Reverse gyrase also displays exquisite sensitivity toward temperature. Raising the reaction temperatures from 80 to 85 degrees C, both of which are within the optimal growth temperature of A. fulgidus, greatly increases enzyme activity for all the supercoiling reactions. For the reaction with AMPPNP, the product is a hypernegatively supercoiled DNA. This dramatic enhancement of the reverse gyrase activity is also correlated with the appearance of DNA in a pre-melting state at 85 degrees C, likely due to the presence of extensively unwound regions in the plasmid. The possible mechanistic insights from these findings will be presented here. 相似文献
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16.
Elastic behavior of RecA-DNA helical filaments 总被引:1,自引:0,他引:1
Escherichia coli RecA protein forms a right-handed helical filament with DNA molecules and has an ATP-dependent activity that exchanges homologous strands between single-stranded DNA (ssDNA) and duplex DNA. We show that the RecA-ssDNA filamentous complex is an elastic helical molecule whose length is controlled by the binding and release of nucleotide cofactors. RecA-ssDNA filaments were fluorescently labelled and attached to a glass surface inside a flow chamber. When the chamber solution was replaced by a buffer solution without nucleotide cofactors, the RecA-ssDNA filament rapidly contracted approximately 0.68-fold with partial filament dissociation. The contracted filament elongated up to 1.25-fold when a buffer solution containing ATPgammaS was injected, and elongated up to 1.17-fold when a buffer solution containing ATP or dATP was injected. This contraction-elongation behavior was able to be repeated by the successive injection of dATP and non-nucleotide buffers. We propose that this elastic motion couples to the elastic motion and/or the twisting rotation of DNA strands within the filament by adjusting their helical phases. 相似文献
17.
P A Lazo 《The Biochemical journal》1985,231(1):185-188
The formation of positive supercoiled DNA by an activity from a hypermutating myeloma line is reported. This activity forms positive supercoils from negative supercoiled DNA, it does not use positive supercoils to form negative ones and does not require an exogenous source of energy. The linking number changes by steps of 1, suggesting a type-I mechanism of action, and there seems to be an upper limit to the degree of positive supercoiling that can be achieved. Positive supercoiled DNA has to be taken into account as a possible structure of DNA in vivo for those functions where torsional stress is involved. 相似文献
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In the yeast Saccharomyces cerevisiae, DNA topoisomerases I and II can functionally substitute for each other in removing positive and negative DNA supercoils. Yeast Delta top1 top2(ts) mutants grow slowly and present structural instability in the genome; over half of the rDNA repeats are excised in the form of extrachromosomal rings, and small circular minichromosomes strongly multimerize. Because these traits can be reverted by the extrachromosomal expression of either eukaryotic topoisomerase I or II, their origin is attributed to the persistence of unconstrained DNA supercoiling. Here, we examine whether the expression of the Escherichia coli topA gene, which encodes the bacterial topoisomerase I that removes only negative supercoils, compensates the phenotype of Delta top1 top2(ts) yeast cells. We found that Delta top1 top2(ts) mutants expressing E. coli topoisomerase I grow faster and do not manifest rDNA excision and minichromosome multimerization. Furthermore, the recombination frequency in repeated DNA sequences, which is increased by nearly two orders of magnitude in Delta top1 top2(ts) mutants relative to the parental TOP+ cells, is restored to normal levels when the bacterial topoisomerase is expressed. These results indicate that the suppression of mitotic hyper-recombination caused by eukaryotic topoisomerases I and II is effected mainly by the relaxation of negative rather than positive supercoils; they also highlight the potential of unconstrained negative supercoiling to promote homologous recombination. 相似文献