Kinetics of Cruciform Formation and Stability of Cruciform Structure in Superhelical DNA

Abstract

This is a study of the kinetics of formation of a cruciform structure from the longest palindromic sequence in plasmid pA03 DNA. DNA was prepared so as to be free of cruciforms even in topoisomers whose negative superhelicity was great enough to induce cruciform formation. Samples of such DNA were incubated at various temperatures, the incubation time varying over a wide range. Then the state was frozen by chilling. Two-dimensional electrophoretic analysis made it possible to estimate the fraction of molecules that got the cruciform structure during incubation. Precautions were taken for electrophoresis conditions to rule out any spontaneous conformational changes within the palindromic region. The relaxation time at the midpoint of the transition ranged from 30 min at 30 C to 50 hrs at 20 C, both in 0.1SSC. An increase in the negative superhelical density by 0.01 led to a 500-fold reduction of the relaxation time at 30 C but had little effect at 20 C. The probability of cruciform formation has been examined as a function of temperature. It has been shown that the cruciform state is no longer the predominant one at elevated temperatures: the cruciformation probability drops to an insignificant value for all of the topoisomers involved. Data have been obtained suggesting that the cruciform formation at the major palindromic site is not the only structural transition possible in pA03 DNA.     

The absence of cruciform structures from pAO3 plasmid DNA in vivo

We extracted pAO3 plasmid DNA from E. coli cells, having "frozen" the transition between cruciform and double-helical conformations in DNA. The characteristic feature of the DNA isolation procedure is that all steps were carried out at temperature between 0 and 4 C and no phenol deproteinization was used, since it has been discovered that phenol destabilizes cruciform structures in pAO3 DNA. Two-dimensional gel electrophoresis has revealed no cruciform structures in the pAO3 DNA preparations obtained this way, although the superhelical density of DNA was sufficient for them. Cruciform structures are absent from intracellular pAO3 DNA at all growth stages of the bacterial culture: stationary and logarithmic, and under the induction of pAO3 DNA replication in chloramphenicol-treated cells.     

Statistical Mechanical Approach for Predicting the Transition to Non-B DNA Structures in Supercoiled DNA

Abstract

Supercoiling causes global twist of DNA structure and the supercoiled state has wide influence on conformational transition. A statistical mechanical approach was made for prediction of the transition probability to non-B DNA structures under torsional stress. A conditional partition function was defined as the sum over all possible states of the DNA sequence with basepair 1 and basepair n being in B-form helix and a recurrence formula was developed which expressed the partition function for basepair n with those for less number of pairs. This new definition permits a quick enumeration of every configuration of secondary structures. Energetic parameters of all conformations concerned, involving B-form, interior loop, cruciform and Z-form, were included in the equation. The probability of transition to each non-B conformation could be derived from these conditional partition functions. For treatment of effects of superhelicity, supercoiling energy was considered, and a twist of each conformation was determined to minimize the supercoiling energy. As the twist itself affects the transition probability, the whole scheme of equations was solved by renormalization technique. The present method permits a simultaneous treatment of serveral types of conformations under a common torsional stress.

A set of energetic parameters of DNA secondary structures has been chosen for calculation. Some DNA sequences were submitted to the calculation, and all the sequences that we submitted gave stable convergence. Some of them have been investigated the critical supercoil density for the transition to non-B DNA structures. Even though the reliability of the set of parameters was not enough, the prediction of secondary structure transition showed good agreement with reported observation. Hence, the present algorithm can estimate the probability of local conformational change of DNA under a given supercoil density, and also be employed to predict some specific sequences in which conformational change is sensitive to superhelicity.     

Long DNA palindromes,cruciform structures,genetic instability and secondary structure repair

Long DNA palindromes pose a threat to genome stability. This instability is primarily mediated by slippage on the lagging strand of the replication fork between short directly repeated sequences close to the ends of the palindrome. The role of the palindrome is likely to be the juxtaposition of the directly repeated sequences by intrastrand base-pairing. This intra-strand base-pairing, if present on both strands, results in a cruciform structure. In bacteria, cruciform structures have proved difficult to detect in vivo, suggesting that if they form, they are either not replicated or are destroyed. SbcCD, a recently discovered exonuclease of Escherichia coli, is responsible for preventing the replication of long palindromes. These observations lead to the proposal that cells may have evolved a post-replicative mechanism for the elimination and/or repair of large DNA secondary structures.     

A Theoretical Study of Formation of DNA Noncanonical Structures Under Negative Superhelical Stress

Abstract

The development of statistical mechanical models of the formation of noncanonical structures in circular DNA and the finding of the energy parameters for these models made it possible to predict the appearance of such structures in a DNA with any given sequence. It does not seem feasible, however, to perform such calculations for DNA sequences of considerable length by allowing for all the possible states. We propose a special algorithm for calculating the thermodynamic characteristics of various conformational rearrangements in DNA that occur under negative supercoilings, allowing for several possible states of each base pair in the chain. Calculations have been performed for a number of natural DNAs. According to these calculations, the most likely noncanonical structures in DNA under normal conditions are cruciform structures and the Z form. The results of the calculations are compared with the experimental data reported in the literature. State diagrams have been computed for a number of inserts in circular DNA that can adopt both the cruciform conformation and the left-handed helical Z form.     

In vitro impact of amino acid-derived bacterial metabolites on colonocyte mitochondrial activity,oxidative stress response and DNA integrity

Background4-hydroxyphenylacetic acid (HO-PAA) is produced by intestinal microbiota from L-tyrosine. High concentrations in human fecal water have been associated with cytotoxicity, urging us to test HO-PAA's effects on human colonocytes. We compared these effects with those of phenylacetic acid (PAA), phenol and acetaldehyde, also issued from amino acids fermentation.MethodsHT-29 Glc^−/+ human colonocytes were exposed for 24 h to metabolites at concentrations between 350 and 1000 μM for HO-PAA and PAA, 250-1500 μM for phenol and 25-500 μM for acetaldehyde. We evaluated metabolites'cytotoxicity with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and DNA quantification assays, reactive oxygen species (ROS) production with H2DCF-DA, and DNA damage with the comet assay. We measured cell oxygen consumption and mitochondrial complexes activity by polarography.ResultsAlthough HO-PAA displayed no cytotoxic effect on colonocytes, it decreased mitochondrial complex I activity and oxygen consumption. This was paralleled by an increase in ROS production and DNA alteration. Cells pretreatment with N-acetylcysteine, a ROS scavenger, decreased genotoxic effects of HO-PAA, indicating implication of oxidative stress in HO-PAA's genotoxicity. PAA and phenol did not reproduce these effects, but were cytotoxic towards colonocytes. Last, acetaldehyde displayed no effect in terms of cytotoxicity and mitochondrial metabolic activity, but increased DNA damage.ConclusionsSeveral bacterial metabolites produced from amino acids displayed deleterious effects on human colonocytes, in terms of genotoxicity (HO-PAA and acetaldehyde) or cytotoxicity (PAA and phenol).General significanceThis study helps understanding the consequences of intestinal microbiota's metabolic activity on the host since amino acids fermentation can lead to the formation of compounds toxic towards colonic epithelial cells.     

Evidence for a large double-cruciform DNA structure on the X chromosome of human and chimpanzee

The human X chromosome consists of a high number of large inverted repeat (IR) DNA sequences which fulfill all requirements for formation of cruciform DNA structures. Such alternative DNA structures are suggested to have a great impact in altering the chromatin architecture and function. Our comprehensive analysis of the corresponding orthologous nucleotide sequences of an IR sequence from Homo sapiens and Pan troglodytes revealed that most of the nucleotide differences between the two species are symmetrical to the apex of the IR, and that the spacer region of the orthologous IRs are in reverse orientation. We provide evidence that this IR forms a large non-B DNA structure containing two Holliday junctions, allowing intrastrand nucleotide pairing of the arms and interstrand pairing of the spacer region of the IR. This structure would extrude into a large double-cruciform DNA structure providing the molecular basis of translocation events and regulation of gene expression. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Localization of a cruciform cutting endonuclease to yeast mitochondria

We have found a cruciform cutting endonuclease in the yeast, Saccharomyces cerevisiae, which localizes to the mitochondria. This activity apparently is associated with the mitochondrial inner membrane since the activity is not released into solution by osmolysis, in contrast to the matrix enzyme, isocitrate dehydrogenase. The cruciform cutting activity appears to be encoded by CCE1. This gene has been shown to encode one of the major cruciform cutting endonucleases present in a yeast cell. In ccel strains, which lack CCE1 endonuclease activity, the mitochondrial cruciform cutting endonucleolytic activity is also absent. Since CCE1 is allelic to MGT1, a gene required for the highly biased transmission of petite mitochondrial DNA in crosses between ⁺ and hypersuppressive ^– cells, it seems likely that the CCE1 endonuclease functions within mitochondria.     

新生婴儿源植物乳杆菌HLPL03的环境耐受性及其代谢功能低聚糖的生物学活性

【目的】解析健康新生婴儿胎便中植物乳杆菌HLPL03的益生功能,评价其环境耐受性及代谢功能低聚糖的生物学活性。【方法】通过耐受胃肠道条件、过氧化氢和抗生素试验,评估植物乳杆菌HLPL03对极端环境的耐受性;利用改良培养基,评价植物乳杆菌HLPL03代谢功能低聚糖的能力;同时,探究功能低聚糖对植物乳杆菌HLPL03抑菌活性、疏水性和黏附能力的影响。【结果】植物乳杆菌HLPL03在pH 2.5条件下培养3 h后,活菌数仍在10⁴ CFU/mL以上;在0.30%胆盐中培养6 h后,活菌数接近10⁷ CFU/mL;在1.0 mmol/L H₂O₂强氧化剂条件下培养6 h,活菌数显著升高(P<0.001);除低聚木糖外,植物乳杆菌HLPL03能代谢多种功能低聚糖,并对常见食源性致病菌具有较强的抑制能力;棉子糖是改善植物乳杆菌HLPL03生物学活性的最佳低聚糖,其能提高菌株表面疏水性达36.1%,且促进菌株在Caco-2细胞上的黏附率由16.78%提高至42.11%。【结论】健康新生婴儿源植物乳杆菌HLPL03具有良好的抗环境胁迫能力,且其生物学活性能被棉子糖等功能低聚糖有效促进,可作为特色乳酸菌进行研究和开发。     

Cellular pathways controlling integron cassette site folding

By mobilizing small DNA units, integrons have a major function in the dissemination of antibiotic resistance among bacteria. The acquisition of gene cassettes occurs by recombination between the attI and attC sites catalysed by the IntI1 integron integrase. These recombination reactions use an unconventional mechanism involving a folded single‐stranded attC site. We show that cellular bacterial processes delivering ssDNA, such as conjugation and replication, favour proper folding of the attC site. By developing a very sensitive in vivo assay, we also provide evidence that attC sites can recombine as cruciform structures by extrusion from double‐stranded DNA. Moreover, we show an influence of DNA superhelicity on attC site extrusion in vitro and in vivo. We show that the proper folding of the attC site depends on both the propensity to form non‐recombinogenic structures and the length of their variable terminal structures. These results draw the network of cell processes that regulate integron recombination.     

Electroantennogram responses of male Costelytra zealandica (Coleoptera: Scarabaeidae) to phenol and related compounds

Electroantennograms recorded from male New Zealand grass grub beetles, Costelytra zealandica (White), were used to compare the activity of phenol with that of closely related phenolics. These compounds had no masking effect on the antennal response. A comparison of activity between phenol and a crude female extract indicated that phenol is the sole pheromone.     

Length-Dependent Cruciform Extrusion in d(GTAC)n Sequences

Abstract

pBR322-derived plasmids have been constructed carrying d(GTAC)_n·d(GTAC)_n inserts of different lengths, in order to investigate the effect of insert size on cruciform extrusion and/or the B-Z transition. Plasmids with n ranging from 4 to 12 are hypersensitive to cleavage by the single-strand specific nucleases, S1 nuclease and Bal31 nuclease. Hypersensitive sites associated with the smaller alternating purine-pyrimidine tracts, however, coexist with the major pBR322 sites. Site-selective cleavage of these plasmids with the resolvase, T7 endonuclease I, demonstrates that all the inserts form cruciform structures when stably integrated into negatively supercoiled plasmids. An increase in the negative superhelical density of the DNA's induces cruciform formation within the insert region, resulting in a reduction in torsional stress consistent with the size of the insert. Moreover, as n decreases, the superhelical density required to stabilise the cruciform state increases. Therefore, the cruciform geometry is the favoured conformation of these d(GTAC)_n·d(GTAC)_n sequences under torsional stress. The stability of these cruciforms increases as n increases, with cruciformation occurring at lower superhelical densities and to the exclusion of the other pBR322 cruciforms.     

Purification and properties of a nuclease from Saccharomyces cerevisiae that cleaves DNA at cruciform junctions

An endonuclease specific for cruciform junctions has been purified from yeast cells treated with a DNA-damaging agent. The activity was followed through five chromatographic steps by assaying for the linearization of supercoiled plasmid DNA, which extrudes cruciform structures in vitro. The sites of cleavage on pColIR215 were sequenced, and nicks were located to positions symmetrically opposed across the cruciform junction. The products of cleavage were unit length linear duplexes that contained terminal hairpin loops. In contrast to pColIR215, the cleavage patterns of pXG540 plasmid DNA were found to be complex, and cuts were found up to 40 bases from an (A-T)34 sequence that extrudes into a cruciform. Little or no activity could be detected on single-stranded DNA, linear duplex DNA, or nicked circular duplex DNA. The nuclease was insensitive to RNase but was inactivated by treatment with proteinase K. Mg2+ was required as cofactor and could not be replaced by Mn2+, Ca2+, Co2+, or Cu2+. The native molecular weight of the activity was approximately 200,000 as estimated by gel filtration.     

Structural and Molecular Biology of the eye Lens Membrane

Abstract

The DNA double helix exhibits local sequence-dependent polymorphism at the level of the single base pair and dinucleotide step. Curvature of the DNA molecule occurs in DNA regions with a specific type of nucleotide sequence periodicities. Negative supercoiling induces in vitro local nucleotide sequence-dependent DNA structures such as cruciforms, left-handed DNA, multistranded structures, etc. Techniques based on chemical probes have been proposed that make it possible to study DNA local structures in cells. Recent results suggest that the local DNA structures observed in vitro exist in the cell, but their occurrence and structural details are dependent on the DNA superhelical density in the cell and can be related to some cellular processes.     

牛源无乳链球菌长尾噬菌体的特性

【目的】分离鉴定噬菌体,对其生物学特性进行研究,并筛选候选毒株为防控牛源无乳链球菌的感染提供依据。【方法】分别采用从牛奶或环境中分离、溶原菌诱导两种方法分离鉴定无乳链球菌噬菌体,利用双层琼脂平板法纯化。将新分离鉴定毒株与前期已分离鉴定的源自乳腺炎牛奶的无乳链球菌噬菌体JX01进行分析和比较,包括噬菌体透射电镜形态观察、对55株无乳链球菌和其他细菌的宿主谱鉴定、噬菌体基因Eco R I、Sal I、Xba I或Pst I的酶切图谱、最适MOI、吸附曲线和一步生长曲线、不同保存条件下的稳定性等。【结果】分离鉴定的3株噬菌体LYGO9、HZ04和p A11(诱导自牛源菌株HAJL2011070601)与JX01比对分析,结果显示,4株噬菌体均为长尾噬菌体;Eco R I、Sal I、Xba I、Pst I的酶切图谱分获4、3、3或2种带型,显示4株噬菌体为不同毒株;均特异性裂解牛源无乳链球菌,对42株牛源无乳链球菌的裂解率如下:LYGO9为28.6%(12/42)、p A11为31%(13/42)、HZ04为47.6%(20/42)、JX01为54.8%(23/42);同时,LYGO9与p A11、HZ04和JX01分别有共同宿主11、12和11株;HZ04与JX01有共同宿主18株,提示它们具有同源性。LYGO9感染宿主的潜伏期短,仅5 min,平均裂解量为30。分离株在SM液中4°C至少可保存1个月。【结论】分离鉴定的3株牛源无乳链球菌噬菌体均为长尾噬菌体,其中LYGO9潜伏期短、裂解量较大。     

微生物燃料电池降解苯酚过程中微电场对阴极微生物多样性的影响

[背景]苯酚废水作为一种毒性强、难降解的废水而备受关注.目前,微生物燃料电池(microbial fuel cell,MFC)已经广泛用于苯酚废水的降解,MFC的产电效果和苯酚的降解效率与反应器内的微生物群落有着密切关系.[目的]为了提高MFC的产电效果及对有害物质的降解能力,需要对MFC中苯酚的降解和微生物群落结构进...     

Nucleosome “phasing” and cruciform structures in circular supercoiled pBR322 DNA

Cruciform structures have been detected in pBR322 supercoiled DNA, both in its naked state and when complexed with histone octamer, using S1 endonuclease cleavage and EcoRI restriction. An inspection of the DNA sequence shows that the S1-hypersensitive sites are very near to AT-rich regions of pBR322 genome. A nucleosome “phasing” in these regions, as found on AT-rich regions of SV40 DNA (15), has been shown by restriction enzymes analysis. On the basis of these results it can be proposed that cruciform structures protrude on the nucleosome surface. This model explains the reason why these structures, which need high superhelical density, can exist in supercoiled DNA partially relaxed by nucleosome formation.