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Phage P1 Cre-loxP site-specific recombination. Effects of DNA supercoiling on catenation and knotting of recombinant products 总被引:5,自引:0,他引:5
Bacteriophage P1 contains a site-specific recombination system consisting of a site, loxP, and a recombinase protein Cre. We have shown that with purified Cre protein we can carry out recombination between two loxP sites in vitro. When that recombination occurs between two sites in direct orientation on the same DNA molecule, we observed the production of free and catenated circular molecules. In this paper we show that recombination between sites in opposite orientation leads to both knotted and unknotted circular products. We also demonstrate that the production of catenanes and knots is influenced by two factors: (1) supercoiling in the DNA substrate, supercoiled DNA substrates yield significantly more catenated and knotted products than nicked circular substrates; and (2) mutations in the loxP site, a class of mutations have been isolated that carry out recombination but result in a distribution of products in which the ratio of catenanes to free circles is increased over that observed with a wild-type site. A more detailed analysis of the products from recombination between wild-type sites indicates: (1) that the catenanes or knots produced by recombination are both simple and complex; (2) that the ratio of free products to catenanes is independent of the distance between the two directly repeated loxP sites; and (3) that for DNA substrates with four loxP sites significant recombination between non-adjacent sites occurs to give free circular products. These observations provide insights into how two loxP sites are brought together during recombination. 相似文献
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Hans V. Westerhoff Mary H. O’Dea Anthony Maxwell Martin Gellert 《Cell biochemistry and biophysics》1988,12(1):157-181
Using purified DNA gyrase to supercoil circular plasmid pBR322 DNA, we examined how the linking number attained at the steady
state (‘static head’) varies with the concentrations of ATP and ADP, both in the absence and presence of spermidine. In the
absence of spermidine at total adenine nucleotide concentrations between 0.35 and 1.4 mM, the static-head linking number was independent of the sum concentration of ATP and ADP, but depended strongly on the ratio
of their concentrations. We established that the same linking number was attained independent of the direction from which
the steady state was approached. The decrease in linking number at static head is more extensive when spermidine is present
in the incubation, but remains a function of the [ATP]-to-[ADP] ratio.
These results are discussed in terms of various kinetic schemes for DNA gyrase. We present one kinetic scheme that accounts
for the experimental observations. According to this scheme our experimental results imply that there is significant slip
in DNA gyrase when spermidine is absent. It is possible that spermidine acts through adjustment of the degree of coupling
of DNA gyrase. 相似文献
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《Cell cycle (Georgetown, Tex.)》2013,12(9):1339-1340
Comment on: Witz G, et al. Proc Natl Acad Sci USA 2011; 108:3608-11. 相似文献
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D M Lilley 《Biochemical Society transactions》1986,14(2):211-213
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DNA supercoiling in vivo 总被引:8,自引:0,他引:8
DNA topoisomerase mutants of Escherichia coli and Saccharomyces cerevisiae were used to study the topological state of intracellular DNA. In E. coli, it is shown that switching off the gene topA encoding DNA topoisomerase I leads to an increase in the degree of negative supercoiling of intracellular DNA and inhibition of the growth of the cells: a d(pCpG)16.d(pCpG)16 sequence on a plasmid is also shown to flip from a right-handed B-helical structure to a left-handed Z-helical structure in vivo when topA is switched off. In S. cerevisiae, the topological state of intracellular DNA is little affected by the cellular levels of the topoisomerases. 相似文献
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Computer simulation of DNA supercoiling 总被引:8,自引:0,他引:8
K V Klenin A V Vologodskii V V Anshelevich A M Dykhne M D Frank-Kamenetskii 《Journal of molecular biology》1991,217(3):413-419
We treat supercoiled DNA within a wormlike model with excluded volume. A modified Monte Carlo approach has been used, which allowed computer statistical-mechanical simulations of moderately and highly supercoiled DNA molecules. Even highly supercoiled molecules do not have a regular shape, though with an increase in writhing the chains look more and more like branched interwound helixes. The averaged writhing (Wr) approximately 0.7 delta Lk. The superhelical free energy F is calculated as a function of the linking number. Lk. The calculations have shown that the generally accepted quadratic dependence of F on Lk is valid for a variety of conditions, though it is by no means universal. Significant deviations from the quadratic dependence are expected at high superhelical density under ionic conditions where the effective diameter of DNA is small. The results are compared with the available experimental data. 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
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DNA supercoiling and prokaryotic transcription 总被引:79,自引:0,他引:79
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C. R. Calladine 《Biopolymers》1980,19(10):1705-1713
Covalently closed circular DNA can exist in different configurations known as circular, toroidal, and interwound. Changes among these forms can be made in several ways, including the insertion of dye molecules between adjacent base pairs, which tends to untwist the double-helical structure. The aim of this paper is to discuss these configurations, and the changes among them, in the context of classical elastomechanics. The concepts of twisting, linkage and writhing are explained. Simple experiments on a twisted linear-elastic rod are described, and it is shown that although the circular and interwound forms may be modeled in this way, the toroidal form does not occur, being mechanically unstable. Theoretical energy calculations by Levitt on bent and twisted DNA show that DNA exhibits a particular kind of nonlinear elasticity in which there is an unusual coupling between bending and twisting. The aim of the paper is to show qualitatively that this special kind of elasticity can stabilize the toroidal form of closed circular DNA. 相似文献
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Baaklini I Usongo V Nolent F Sanscartier P Hraiky C Drlica K Drolet M 《Journal of bacteriology》2008,190(22):7346-7356
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The packing of DNA inside bacteriophages arguably yields the simplest example of genome organization in living organisms. As an assay of packing geometry, the DNA knot spectrum produced upon release of viral DNA from the P4 phage capsid has been analyzed, and compared to results of simulation of knots in confined volumes. We present new results from extensive stochastic sampling of confined self-avoiding and semiflexible circular chains with volume exclusion. The physical parameters of the chains (contour length, cross section, and bending rigidity) have been set to match those of P4 bacteriophage DNA. By using advanced sampling techniques, involving multiple Markov chain pressure-driven confinement combined with a thermodynamic reweighting technique, we establish the knot spectrum of the circular chains for increasing confinement up to the highest densities for which available algorithms can exactly classify the knots. Compactified configurations have an enclosing hull diameter ∼2.5 times larger than the P4 caliper size. The results are discussed in relation to the recent experiments on DNA knotting inside the capsid of a P4 tailless mutant. Our investigation indicates that confinement favors chiral knots over achiral ones, as found in the experiments. However, no significant bias of torus over twist knots is found, contrary to the P4 results. The result poses a crucial question for future studies of DNA packaging in P4: is the discrepancy due to the insufficient confinement of the equilibrium simulation or does it indicate that out-of-equilibrium mechanisms (such as rotation by packaging motors) affect the genome organization, hence its knot spectrum in P4? 相似文献
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Nucleoids isolated from Escherichia coli strains carrying temperature-sensitive gyrA or gyrB mutations were examined by sedimentation in ethidium bromide-containing sucrose density gradients. A shift to restrictive temperature resulted in nucleoid DNA relaxation in all of the mutant strains. Three of these mutants exhibited reversible nucleoid relaxation: when cultures incubated at restrictive temperature were cooled to 0 degree C over a 4- to 5-min period, supercoiling returned to levels observed with cells grown at permissive temperature. Incubation of these three mutants at restrictive temperature also caused nucleoid sedimentation rates to increase by about 50%. 相似文献
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L M Fisher C A Austin R Hopewell E E Margerrison M Oram S Patel K Plummer J H Sng S Sreedharan 《Philosophical transactions of the Royal Society of London. Series B, Biological sciences》1992,336(1276):83-91
Bacterial DNA gyrase and the eukaryotic type II DNA topoisomerases are ATPases that catalyse the introduction or removal of DNA supercoils and the formation and resolution of DNA knots and catenanes. Gyrase is unique in using ATP to drive the energetically unfavourable negative supercoiling of DNA, an example of mechanochemical coupling: in contrast, eukaryotic topoisomerase II relaxes DNA in an ATP-requiring reaction. In each case, the enzyme-DNA complex acts as a 'gate' mediating the passage of a DNA segment through a transient enzyme-bridged double-strand DNA break. We are using a variety of genetic and enzymic approaches to probe the nature of these complexes and their mechanism of action. Recent studies will be described focusing on the role of DNA wrapping on the A2B2 gyrase complex, subunit activities uncovered by using ATP analogues and the coumarin and quinolone inhibitors, and the identification and functions of discrete subunit domains. Homology between gyrase subunits and the A2 homodimer of eukaryotic topo II suggests functional conservation between these proteins. The role of ATP hydrolysis by these topoisomerases will be discussed in regard to other energy coupling systems. 相似文献
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Archaeal histones have significant sequence and structural similarity to their eukaryal counterparts. However, whereas DNA is wrapped in negatively constrained supercoils in eukaryal nucleosomes, it has been reported that DNA is positively supercoiled by archaeal nucleosomes. This was inferred from experiments performed at low temperature and low salt concentrations, conditions markedly different from those expected for many archaea in vivo. Here, we report that the archaeal histones HMf and HTz wrap DNA in negatively constrained supercoils in buffers containing potassium glutamate (K-Glu) above 300 mM, either at 37 degrees C or at 70 degrees C. This suggests that high salt concentrations allow an alternate archaeal nucleosome topology: a left-handed tetramer rather than the right-handed tetramer seen in low salt conditions. In contrast, the archaeal histone MkaH produces DNA negative supercoiling at all salt concentrations, suggesting that this duality of structure is not possible for this atypical protein, which is formed by the association of two histone folds in a single polypeptide. These results extend the already remarkable similarity between archaeal and eukaryal nucleosomes, as it has been recently shown that DNA can be wrapped into either positive or negative supercoils around the H3/H4 tetramer. Negative supercoiling could correspond to the predominant physiological mode of DNA supercoiling in archaeal nucleosomes. 相似文献
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DNA supercoiling and eukaryotic transcription--cause and effect 总被引:1,自引:0,他引:1