Peroxiredoxin 6 modulates Toll signaling pathway and protects DNA damage against oxidative stress in red swamp crayfish (Procambarus clarkii).

Peroxiredoxin 6 (Prx6) is an important member of the peroxiredoxin family that plays critical roles in protecting host against the toxicity of oxidative stress and participates in cell signaling. Herein, we report Prx6 gene from red swamp crayfish, Procambarus clarkii. The cDNA fragment of PcPrx6 was 660 bp, encoding a 219 amino acid residues protein. The quantitative real time PCR analysis showed ubiquitous expression of PcPrx6 mRNA in the tested tissues. The challenge with peptidoglycan and Poly I:C remarkably suppressed the mRNA level of PcPrx6 in hepatopancreas at 3, 12, 48 h compared with the PBS control. However, the expression level significantly increased after 36 h of their treatment. The knockdown of PcPrx6 by small interference RNA significantly enhanced the transcript levels of Toll pathway-responsive genes at 24 h. Recombinant PcPrx6 protein was purified using affinity chromatography and analyzed for its biological role. The results revealed that the recombinant PcPrx6 protein manifested the ability to protect supercoiled DNA damage from oxidative stress elicited by mixed function oxidative assay. Altogether, PcPrx6 may have multiple functional roles in the physiology of P. clarkii, since it negatively regulates the Toll signaling transduction and protects supercoiled DNA damage from oxidative stress.

Mechanical bounds to transcriptional noise.

Over the past several decades it has been increasingly recognized that stochastic processes play a central role in transcription. Although many stochastic effects have been explained, the source of transcriptional bursting (one of the most well-known sources of stochasticity) has continued to evade understanding. Recent results have pointed to mechanical feedback as the source of transcriptional bursting, but a reconciliation of this perspective with preexisting views of transcriptional regulation is lacking. In this article, we present a simple phenomenological model that is able to incorporate the traditional view of gene expression within a framework with mechanical limits to transcription. By introducing a simple competition between mechanical arrest and relaxation copy number probability distributions collapse onto a shared universal curve under shifting and rescaling and a lower limit of intrinsic noise for any mean expression level is found.

The importance of being supercoiled: how DNA mechanics regulate dynamic processes.

Through dynamic changes in structure resulting from DNA-protein interactions and constraints given by the structural features of the double helix, chromatin accommodates and regulates different DNA-dependent processes. All DNA transactions (such as transcription, DNA replication and chromosomal segregation) are necessarily linked to strong changes in the topological state of the double helix known as torsional stress or supercoiling. As virtually all DNA transactions are in turn affected by the torsional state of DNA, these changes have the potential to serve as regulatory signals detected by protein partners. This two-way relationship indicates that DNA dynamics may contribute to the regulation of many events occurring during cell life. In this review we will focus on the role of DNA supercoiling in the cellular processes, with particular emphasis on transcription. Besides giving an overview on the multiplicity of factors involved in the generation and dissipation of DNA torsional stress, we will discuss recent studies which give new insight into the way cells use DNA dynamics to perform functions otherwise not achievable. This article is part of a Special Issue entitled: Chromatin in time and space.

SCF(Dia2) regulates DNA replication forks during S-phase in budding yeast.

Dia2 is an F-box protein, which is involved in the regulation of DNA replication in the budding yeast Saccharomyces cerevisiae. The function of Dia2, however, remains largely unknown. In this study, we report that Dia2 is associated with the replication fork and regulates replication fork progression. Using modified yeast two-hybrid screening, we have identified components of the replisome (Mrc1, Ctf4 and Mcm2), as Dia2-binding proteins. Mrc1 and Ctf4 were ubiquitinated by SCF(Dia2) both in vivo and in vitro. Domain analysis of Dia2 revealed that the leucine-rich repeat motif was indispensable for the regulation of replisome progression, whereas the tetratricopeptide repeat (TPR) motif was involved in the interaction with replisome components. In addition, the TPR motif was shown to be involved in Dia2 stability; deleting the TPR stabilized Dia2, mimicking the effect of DNA damage. ChIP-on-chip analysis illustrated that Dia2 localizes to the replication fork and regulates fork progression on hydroxyurea treatment. These results demonstrate that Dia2 is involved in the regulation of replisome activity through a direct interaction with replisome components.

Supercoiled Minivector DNA resists shear forces associated with gene therapy delivery.

Supercoiled DNAs varying from 281 to 5302 bp were subjected to shear forces generated by aerosolization or sonication. DNA shearing strongly correlated with length. Typical sized plasmids (≥ 3000 bp) degraded rapidly. DNAs 2000-3000 bp persisted ~10 min. Even in the absence of condensing agents, supercoiled DNA <1200 bp survived nebulization, and increased forces of sonication were necessary to shear it. Circular vectors were considerably more resistant to shearing than linear vectors of the same length. DNA supercoiling afforded additional protection. These results show the potential of shear-resistant Minivector DNAs to overcome one of the major challenges associated with gene therapy delivery.

Quantitative determination of the infectivity of the proviral DNA of a retrovirus in vitro: Evaluation of methods for DNA inactivation.

All viral vaccines contain contaminating residual DNA derived from the production cell substrate. The potential risk of this DNA, particularly when derived from tumorigenic cells, has been debated for over 40 years. While the major risk has been considered to be the oncogenicity of the DNA, another potential risk is that a genome of an infectious virus is present in this DNA. Such a genome might generate an infectious agent that could establish an infection in vaccine recipients. To determine the quantity of a retroviral provirus in cellular DNA that can establish a productive infection in vitro, we developed a transfection/co-culture system capable of recovering infectious virus from 1 pg of cloned HIV DNA and from 2 microg of cellular DNA from HIV-infected cells. We demonstrate that infectivity can be reduced to below detectable levels either by lowering the median size of the DNA to 350 base pairs or by treatment with beta-propiolactone. From the amount of reduction of infectivity, we calculate that clearance values in excess of 10(7) are attainable with respect to the infectivity associated with residual cell-substrate DNA. Thus, the potential risk associated with DNA can be substantially reduced by degradation or by chemical inactivation.

DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites.

The tumour suppressor protein p53 is one of the most important factors regulating cell proliferation, differentiation and programmed cell death in response to a variety of cellular stress signals. P53 is a nuclear phosphoprotein and its biochemical function is closely associated with its ability to bind DNA in a sequence-specific manner and operate as a transcription factor. Using a competition assay, we investigated the effect of DNA topology on the DNA binding of human wild-type p53 protein. We prepared sets of topoisomers of plasmid DNA with and without p53 target sequences, differing in their internal symmetry. Binding of p53 to DNA increased with increasing negative superhelix density (-sigma). At -sigma < or = 0.03, the relative effect of DNA supercoiling on protein-DNA binding was similar for DNA containing both symmetrical and non-symmetrical target sites. On the other hand, at higher -sigma, target sites with a perfect inverted repeat sequence exhibited a more significant enhancement of p53 binding as a result of increasing levels of negative DNA supercoiling. For -sigma = 0.07, an approx. 3-fold additional increase in binding was observed for a symmetrical target site compared with a non-symmetrical target site. The p53 target sequences possessing the inverted repeat symmetry were shown to form a cruciform structure in sufficiently negative supercoiled DNA. We show that formation of cruciforms in DNA topoisomers at -sigma > or = 0.05 correlates with the extra enhancement of p53-DNA binding.

Bending and supercoiling of DNA at the attachment site of bacteriophage lambda.

Integration of the DNA of bacteriophage lambda into the chromosome of E. coli depends on the formation of a complex nucleoprotein array at a specific locus on the phage genome, the attachment site. Recent work shows how bending of this DNA (induced by a specific DNA-binding protein), and strain in this DNA (induced by supercoiling) contribute to the formation of the nucleoprotein structure. Further, there are new insights into the way this structure directs critical events during recombination.

DNA supercoiling - a global transcriptional regulator for enterobacterial growth?

A fundamental principle of exponential bacterial growth is that no more ribosomes are produced than are necessary to support the balance between nutrient availability and protein synthesis. Although this conclusion was first expressed more than 40 years ago, a full understanding of the molecular mechanisms involved remains elusive and the issue is still controversial. There is currently agreement that, although many different systems are undoubtedly involved in fine-tuning this balance, an important control, and in our opinion perhaps the main control, is regulation of the rate of transcription initiation of the stable (ribosomal and transfer) RNA transcriptons. In this review, we argue that regulation of DNA supercoiling provides a coherent explanation for the main modes of transcriptional control - stringent control, growth-rate control and growth-phase control - during the normal growth of Escherichia coli.

Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro.

We present evidence for the existence of prolactin upstream factor 1 (PUF-1) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived GH3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. GH3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28-bp region (-63 to -36)which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed.

Characterization of the repressed 5S DNA minichromosomes assembled in vitro with a high-speed supernatant of Xenopus laevis oocytes.

We describe an in vitro system, based on the Xenopus laevis oocyte supernatant of Glikin et al. (G. Glikin, I. Ruberti, and A. Worcel, Cell 37:33-41, 1984), that packages DNA into minichromosomes with regularly spaced nucleosomes containing histones H3, H4, H2A, and H2B but no histone H1. The same supernatant also assembles the 5S RNA transcription complex; however, under the conditions that favor chromatin assembly, transcription is inhibited and a phased nucleosome forms over the 5S RNA gene. The minichromosomes that are fully loaded with nucleosomes remain refractory to transcriptional activation by 5S RNA transcription factors. Our data suggest that this repression is caused by a nucleosome covering the 5S RNA gene and that histone H1 is not required for regular nucleosome spacing or for gene repression in this system.

DNA supercoiling and transcription control: a model from the study of suppression of the leu-500 mutation in Salmonella typhimurium topA- strains.

DNA supercoiling is known to modulate gene expression. The functional relationship between DNA supercoiling and transcription initiation has been established genetically and biochemically. The molecular mechanism whereby DNA supercoiling regulates gene expression remains unclear however. Quite commonly, the same gene responds to the same DNA supercoiling change differently when the gene is positioned at different locations. Such strong positional effects on gene expression suggest that rather than the overall DNA supercoiling change, the variation of DNA supercoiling at a local site might be important for transcription control. We have started to understand the local DNA supercoiling dynamic on the chromosome. As a primary source of local DNA supercoiling fluctuation, transcription-driven DNA supercoiling is important in determining the chromosome supercoiling dynamic and theoretically, therefore, for transcription control as well. Indeed, by studying the coordinated expression of genes in the ilvIH-leuO-leuABCD gene cluster, we found that transcription-driven DNA supercoiling governs the expression of a group of functionally related genes in a sequential manner. Based on the findings in this model system, we put forward the possible mechanisms whereby DNA supercoiling plays its role in transcription control.

DNA polymerase alpha-primase complexes from carcinogen-treated Chinese hamster ovary cells.

In order to investigate whether carcinogens induce alterations of the DNA polymerase alpha-primase complex we compared the physiochemical and catalytic properties and the fidelity of DNA synthesis of DNA polymerase alpha-primase complexes from carcinogen-treated and untreated Chinese hamster ovary cells. Complexes were purified by ion exchange or by immunoaffinity chromatography and both DNA-polymerizing activities and those of ancillary enzymes, such as RNA primase and exonuclease, were examined. Further characterization of the complexes included determination of the relative molecular masses, sedimentation coefficients, and diffusion coefficients, and measurements of the KmS for deoxynucleotide triphosphates and DNA templates, which were identical for the preparations from both carcinogen-treated and untreated cells. The fidelity of DNA polymerase alpha-primase complexes measured by the phi X174am3 reversion assay was also similar in carcinogen-treated and untreated cells. Thus, a carcinogen-mediated induction of a DNA polymerase alpha-primase complex with low fidelity was not observed within the detection limits of the phi X174 assay. RNA primase was found to be an ancillary enzyme activity of the DNA polymerase alpha from both carcinogen-treated and untreated cells; however, the RNA primase:DNA polymerase alpha activity ratio was significantly higher in DNA polymerase alpha-primase complexes from carcinogen-treated cells. These complexes also exhibited an at least 3 times greater velocity of synthesis with supercoiled or unprimed single-stranded DNAs as templates. Since the binding sites of DNA polymerase alpha-primase complexes for deoxynucleotide triphosphates and DNA templates were shown to be identical before and after treatment of cells with carcinogens (i.e., identical Km values for different DNA templates and Ki values for specific inhibitors), the increased synthesis catalyzed by the DNA polymerase alpha-primase complex from carcinogen-treated cells might be due to a carcinogen-induced alteration of an accessory protein of the complex.

Chromatin assembly on plasmid DNA in vitro. Apparent spreading of nucleosome alignment from one region of pBR327 by histone H5.

We have found that histone H5 (or H1) induces physiological nucleosome spacings and extensive ordering on some plasmid constructions, but not on others, in a fully defined in vitro system. Plasmid pBR327 containing DNA insertions with lengths close to 300 base-pairs permitted histone H5 to induce a remarkable degree of nucleosome alignment. Seventeen multiples of a unit 210(+/- 4) base-pair repeat, covering the entire plasmid, were detected. Plasmid pBR327, not containing a DNA insert, permitted continuous alignment of only a few nucleosomes. These observations suggest that a necessary requirement in this system for histone H5 (or H1)-induced nucleosome alignment on small (less than 4 kb; 1 kb = 10(3) bases or base-pairs) circular plasmids may be that the total DNA length must be close to an integer multiple of the nucleosome repeat length generated, a type of boundary effect. Consistent with this hypothesis, five deletion constructs of pBR327 (not containing inserts), that spanned 64% of the plasmid, and possessed DNA lengths close to integer multiples of 210 base-pairs, permitted nucleosome alignment by histone H5. We have also found that plasmid length adjustment is not a sufficient condition for nucleosome alignment. For example, plasmids pBR322 and pUC18 did not permit nucleosome alignment when adjusted to near-integer multiples of 210 base-pairs. Also, for pBR327 that contained a length-adjusted deletion in one particular region, appreciable nucleosome alignment no longer occurred. These data suggest that a contiguous approximately 800 base-pair region of pBR327, interrupted in pBR322 and not present in pUC18, can nucleate histone H5-induced nucleosome alignment, which can then spread to adjacent chromatin. Supporting this idea, a positioned five-nucleosome array appears to originate in the required region. Additionally, on a larger (6.9 kb) plasmid construction, the "chromatin organizing region" of pBR327 and adjacent DNA on one side of it exhibited preferred H5-induced nucleosome alignment.

Correlation between the DNA supercoiling and the initiation of transcription by Escherichia coli RNA polymerase in vitro: role of the sequences upstream of the promoter region.

Binding of Escherichia coli RNA polymerase and the abortive initiation of transcription at the A2 promoter of bacteriophage T7, separately cloned in pBR322, was found to be strongly dependent on the degree of supercoiling of the plasmid. Supercoiling does not seem to play any role in the initiation of transcription at the T7A1 promoter under identical conditions. Plasmid containing T7A2 promoter was found to be less amenable to S1 nuclease in comparison to that having T7A1. Sequence comparison reveals a high G/C content upstream to the -35 region of T7A2 which by extra duplex stability probably renders the initiation of transcription more dependent on the state of supercoiling of the template.

Age-related changes of DNA winding and repair in human peripheral lymphocytes.

Superhelical density and incision capacity for UV light-induced damage have been studied in nuclear DNA of human peripheral lymphocytes as a function of donor age. With advancing age between 20 and 90 years the content of negative superhelical turns increases, whereas the ability to incise UV lesions is impaired. These data may be suggestive of an immature lymphoid cell state arising in aging.

DNA supercoiling and transcription in Escherichia coli: The FIS connection.

The nucleoid-associated protein FIS modulates the topology of DNA in a growth-phase dependent manner functioning homeostatically to counteract excessive levels of negative superhelicity. We propose that this is achieved by at least two mechanisms: the physical constraint of low levels of negative superhelicity by FIS binding to DNA and by a reduction in the expression and effectiveness of DNA gyrase. In addition, high levels of expression of the fis gene do themselves require a high negative superhelical density. On DNA substrates containing phased high affinity binding sites, as exemplified by the upstream activating sequence of the tyrT promoter, FIS forms tightly bent DNA structures, or microloops, that are necessary for the optimal expression of the promoter. We suggest that these microloops compensate in part for the FIS-induced lowering of the superhelical density.

Transcription-driven DNA supercoiling and gene expression control.

DNA supercoiling plays important roles in gene expression regulation, although, the underlying mechanisms whereby DNA supercoiling modulates gene expression remain elusive. The fact that the transcription process itself generates DNA supercoiling has further complicated the issue. Transcription-driven DNA supercoiling is local and transient. Such a DNA supercoiling effect is likely to play important roles in controlling complex gene expression regulation. Using the suppression of the leu-500 mutation in Salmonella typhimurium topA mutants as a model system, we put forward our view of the effects of transcription-driven DNA supercoiling on gene expression control.

Radiation sensitivity correlates with changes in DNA supercoiling and nucleoid protein content in cells of three Chinese hamster cell lines.

We have investigated the composition of nuclear matrix proteins and DNA supercoiling characteristics of cell lines expressing altered radiation sensitivity. Chinese hamster ovary cell lines 4364 (wild-type), XR-1 (DSB repair-deficient, radiosensitive) and XR-122 (a radioresistant variant of XR-1 bearing human chromosome 5) were used as a model to study the relationship between intrinsic radiation sensitivity and the level of DNA supercoiling ability within chromatin loops and the composition of nuclear matrix proteins. Analysis of the ability of DNA loop domains to undergo changes in DNA supercoiling in the presence of DNA damage revealed that the degree of inhibition of loop rewinding was greater in the radiation-sensitive cells (XR-1) compared to the radiation-resistant cells (4364 and XR-122). Furthermore, the loop-rewinding characteristics correlated inversely with the clonogenic survival of these cells after exposure to ionizing radiation. Since DNA loops are anchored to the nuclear matrix by protein-DNA anchor points, a study of the nuclear matrix proteins by high-resolution 2D-PAGE was conducted for these cells to determine whether differential inhibition of loop rewinding could be due to differences in the DNA loop-protein anchor points in these cells. The XR-1 cells showed an overall absence of 13 proteins compared to the 4364 cells. Of these 13, 5 were restored in XR-122 cells. These results are consistent with the hypothesis that stability of the DNA loop domains in the presence of DNA damage contributes to the expression of potentially lethal damage by ionizing radiation.

Effect of the potential triplex DNA region on the in vitro expression of bacterial beta-lactamase gene in superhelical recombinant plasmids.

The effect of a pyrimidine/purine-biased stretch which has the potential to form an unusual triplex DNA structure on gene expression has been analyzed by measuring the activity of beta-lactamase as a reporter gene in recombinant plasmids. The Escherichia coli transformant carrying the plasmid p7ERS which has a potential triplex DNA region expressed about twofold more beta-lactamase activity than that carrying the plasmid pUC19. Since the expression of beta-lactamase has been shown to be affected by template topology in vitro, this in vivo observation suggests that the inserted pyrimidine/purine-biased stretch modulates the topology of flanking regions by forming unusual DNA structure to keep the template at the superhelicity favorable for the expression of beta-lactamase.