Use of double-stranded DNA mini-circles to characterize the covalent topoisomerase-DNA complex.

The enzymatic DNA relaxation requires the DNA to be transiently nicked and rejoined, the covalent topoisomerase-DNA complex being a key intermediate of the nicking-joining reaction. Practically, this reaction is most often characterized by oligonucleotides. However, the incision-religation of an oligonucleotide does not fully recapitulate the incision-religation occuring during relaxation and the preferred substrate for such reaction characterization is supercoiled DNA. We therefore developed a method that used radiolabeled supercoiled DNA mini-circles to characterize the covalent enzyme-DNA complex formed during a relaxation reaction. Resolution of the relaxation products under different conditions permitted to quantify the proportion of covalent complex formed during the relaxation catalyzed by two topoisomerase models, the Escherichia coli topoisomerase I and the calf thymus topoisomerase I. As expected, the covalent complex formed with the calf thymus topoisomerase I was significantly enriched by camptothecin, a widely-used inhibitor of this topoisomerase, and a salt jump permitted the multiple topoisomerases trapped per mini-circle to complete the reaction cycle. The identified positions of the camptothecin-induced incision sites were shown to be independent of the linking number and the substrate circular nature Overall, our results demonstrate that supercoiled mini-circles constitute a powerful and polyvalent substrate to characterize the mechanism of action of novel topoisomerases and inhibitors, including the incision-religation reaction.

Construction of DNA hemicatenanes from two small circular DNA molecules.

DNA hemicatenanes, one of the simplest possible junctions between two double stranded DNA molecules, have frequently been mentioned in the literature for their possible function in DNA replication, recombination, repair, and organization in chromosomes. They have been little studied experimentally, however, due to the lack of an appropriate method for their preparation. Here we have designed a method to build hemicatenanes from two small circular DNA molecules. The method involves, first, the assembly of two linear single strands and their circularization to form a catenane of two single stranded circles, and, second, the addition and base-pairing of the two single stranded circles complementary to the first ones, followed by their annealing using DNA topoisomerase I. The product was purified by gel electrophoresis and characterized. The arrangement of strands was as expected for a hemicatenane and clearly distinct from a full catenane. In addition, each circle was unwound by an average of half a double helical turn, also in excellent agreement with the structure of a hemicatenane. It was also observed that hemicatenanes are quickly destabilized by a single cut on either of the two strands passing inside the junction, strongly suggesting that DNA strands are able to slide easily inside the hemicatenane. This method should make it possible to study the biochemical properties of hemicatenanes and to test some of the hypotheses that have been proposed about their function, including a possible role for this structure in the organization of complex genomes in loops and chromosomal domains.

Expression of Epstein-Barr virus EBNA1 protein in Escherichia coli: purification under nondenaturing conditions and use in DNA-binding studies.

Epstein-Barr virus nuclear antigen 1 (EBNA1) is a viral protein required for stable replication and segregation of DNA episomes containing the Epstein-Barr virus (EBV) origin of replication, OriP. Overproduction of EBNA1 protein in Escherichia coli has previously been shown to be difficult due to the large number of codons in EBNA1 gene that are infrequently used in E. coli. Here we changed the 26 rare codons that are found among the first 78 codons of EBNA1 gene, and replaced them with codons that encode the same amino-acids but are abundant in E. coli. This led to a significant improvement of EBNA1 expression in a standard E. coli strain. Partial EBNA1 polypeptides of 11.5-16 kDa extending from the N-terminus to the second arginine and glycine-rich region were extremely abundant in the extract, however, resulting in a second limitation of the level of EBNA1 expression. EBNA1 was expressed as a fusion with a C-terminal six-histidine tag in order to get rid of the short polypeptides by Ni-NTA affinity purification, and salt conditions were used that allowed us to extract and purify EBNA1 without resorting to protein denaturing reagents. The purified protein was used in DNA-binding experiments with DNA fragments containing specific EBNA1 sites. The E. coli-expressed protein formed specific DNA-protein complexes that could be analyzed in polyacrylamide gels without showing the aggregation, or linking, phenomenon that is usually observed with EBNA1 expressed in eukaryotic cells. EBNA1 protein expressed in E. coli should therefore prove useful to further study the biochemical properties of this crucial Epstein-Barr virus protein.

A high-sensitivity method for detection and measurement of HMGB1 protein concentration by high-affinity binding to DNA hemicatenanes.

BACKGROUND: Protein HMGB1, an abundant nuclear non-histone protein that interacts with DNA and has an architectural function in chromatin, was strikingly shown some years ago to also possess an extracellular function as an alarmin and a mediator of inflammation. This extracellular function has since been actively studied, both from a fundamental point of view and in relation to the involvement of HMGB1 in inflammatory diseases. A prerequisite for such studies is the ability to detect HMGB1 in blood or other biological fluids and to accurately measure its concentration. METHODOLOGY/PRINCIPAL FINDINGS: In addition to classical techniques (western blot, ELISA) that make use of specific anti-HMGB1 antibodies, we present here a new, extremely sensitive technique that is based on the fact that hemicatenated DNA loops (hcDNA) bind HMGB1 with extremely high affinity, higher than the affinity of specific antibodies, similar in that respect to DNA aptamers. DNA-protein complexes formed between HMGB1 and radiolabeled hcDNA are analyzed by electrophoresis on nondenaturing polyacrylamide gels using the band-shift assay method. In addition, using a simple and fast protocol to purify HMGB1 on the basis of its solubility in perchloric acid allowed us to increase the sensitivity by suppressing any nonspecific background. The technique can reliably detect HMGB1 at a concentration of 1 pg per microliter in complex fluids such as serum, and at much lower concentrations in less complex samples. It compares favorably with ELISA in terms of sensitivity and background, and is less prone to interference from masking proteins in serum. CONCLUSION: The new technique, which illustrates the potential of DNA nanoobjects and aptamers to form high-affinity complexes with selected proteins, should provide a valuable tool to further investigate the extracellular functions of HMGB1 and its involvement in inflammatory pathologies.

HMGB1 interacts with human topoisomerase IIalpha and stimulates its catalytic activity.

DNA topoisomerase IIalpha (topo IIalpha) is an essential nuclear enzyme and its unique decatenation activity has been implicated in many aspects of chromosome dynamics such as chromosome replication and segregation during mitosis. Here we show that chromatin-associated protein HMGB1 (a member of the large family of HMG-box proteins with possible functions in DNA replication, transcription, recombination and DNA repair) promotes topo IIalpha-mediated catenation of circular DNA, relaxation of negatively supercoiled DNA and decatenation of kinetoplast DNA. HMGB1 interacts with topo IIalpha and this interaction, like the stimulation of the catalytic activity of the enzyme, requires both HMG-box domains of HMGB1. A mutant of HMGB1, which cannot change DNA topology stimulates DNA decatenation by topo IIalpha indistinguishably from the wild-type protein. Although HMGB1 stimulates ATP hydrolysis by topo IIalpha, the DNA cleavage is much more enhanced. The observed abilities of HMGB1 to interact with topo IIalpha and promote topo IIalpha binding to DNA suggest a mechanism by which HMGB1 stimulates the catalytic activity of the enzyme via enhancement of DNA cleavage.

High-mobility group box 1 protein induces HIV-1 expression from persistently infected cells.

BACKGROUND: Necrosis is a frequent condition during AIDS, notably in organs targetted by opportunistic infections. Soluble factors released by necrotic cells are important for signalling cell damage, but little is known concerning their effect on HIV-1 replication. We focused on HMGB1, an abundant component of the chromatin that is released from necrotic cells and can act as a pro-inflammatory mediator. MATERIALS AND METHODS: A native form of HMGB1 was obtained from necrotic Hela cells, whereas a purified recombinant HMGB1 was generated in Escherichia coli. ACH-2 and U1 cells were used as models of persistent HIV-1 infection in lymphocytes and monocytes. Reactivation from latency was also investigated ex vivo using peripheral blood mononuclear cells (PBMC) collected from HIV-1-infected patients controlled by HAART. HIV-1 expression was quantified by enzyme-linked immunosorbent assay, real-time reverse transcription-polymerase chain reaction and branched DNA techniques. Flow cytometry and blocking experiments were used to identify the receptor used by HMGB1. Chromatin immunoprecipitation was used to investigate long-terminal repeat activation upon stimulation by HMGB1. RESULTS: HMGB1 increased HIV-1 transcription in chronically infected cells, a process that did not require de-novo protein synthesis. HIV-1 induction relied on HMGB1 interaction with the receptor for advanced glycation end-products. The activation pathway involved p38 and extracellular signal-related kinase as well as nuclear factor kappa B binding to the HIV-1 promoter. Finally, HMGB1 reactivated HIV-1 from latently infected PBMC collected in aviraemic HIV-infected patients. CONCLUSION: This work establishes for the first time a link between necrosis and HIV-1 replication, which involves HMGB1, a soluble mediator released by damaged cells.

Determinants of specific binding of HMGB1 protein to hemicatenated DNA loops.

Protein HMGB1 has long been known as one of the most abundant non-histone proteins in the nucleus of mammalian cells, and has regained interest recently for its function as an extracellular cytokine. As a DNA-binding protein, HMGB1 facilitates DNA-protein interactions by increasing the flexibility of the double helix, and binds specifically to distorted DNA structures. We have previously observed that HMGB1 binds with extremely high affinity to a novel DNA structure, hemicatenated DNA loops (hcDNA), in which double-stranded DNA fragments containing a tract of poly(CA).poly(TG) form a loop maintained at its base by a hemicatenane. Here, we show that the single HMGB1 domains A and B, the HMG-box domain of sex determination factor SRY, as well as the prokaryotic HMGB1-like protein HU, specifically interact with hcDNA (Kd approximately 0.5 nM). However, the affinity of full-length HMGB1 for hcDNA is three orders of magnitude higher (Kd<0.5 pM) and requires the simultaneous presence of both HMG-box domains A and B plus the acidic C-terminal tail on the molecule. Interestingly, the high affinity of the full-length protein for hcDNA does not decrease in the presence of magnesium. Experiments including a comparison of HMGB1 binding to hcDNA and to minicircles containing the CA/TG sequence, binding studies with HMGB1 mutated at intercalating amino acid residues (involved in recognition of distorted DNA structures), and exonuclease III footprinting, strongly suggest that the hemicatenane, not the DNA loop, is the main determinant of the affinity of HMGB1 for hcDNA. Experiments with supercoiled CA/TG-minicircles did not reveal any involvement of left-handed Z-DNA in HMGB1 binding. Our results point to a tight structural fit between HMGB1 and DNA hemicatenanes under physiological conditions, and suggest that one of the nuclear functions of HMGB1 could be linked to the possible presence of hemicatenanes in the cell.

High-affinity binding of tumor-suppressor protein p53 and HMGB1 to hemicatenated DNA loops.

We have recently observed that chromatin architectural protein HMGB1 (previously reported to be involved in numerous biological processes such as DNA replication, recombination, repair, tumor growth, and metastasis) could bind with extremely high affinity (K(d) < 1 pM) to a novel DNA structure that forms a DNA loop maintained at its base by a hemicatenane (hcDNA). The loop of hcDNA contains a track of repetitive sequences derived from CA-microsatellites. Here, we report using a gel-retardation assay that tumor-suppressor protein p53 can also bind to hcDNA. p53 is a crucial molecule protecting cells from malignant transformation by regulating cell-cycle progression, apoptosis, and DNA repair by activation or repression of transcription of its target genes by binding to specific p53 DNA-binding sites and/or certain types of DNA lesions or alternative DNA structures. The affinity of p53 for hcDNA (containing sequences with no resemblance to the p53 DNA consensus sequence) is >40-fold higher (K(d) approximately 0.5 nM) than that for its natural specific binding sites within its target genes (Mdm2 promoter). Binding of p53 to hcDNA remains detectable in the presence of up to approximately 4 orders of magnitude of mass excess of competitor linear DNA, suggesting a high specificity of the interaction. p53 displays a higher affinity for hcDNA than for DNA minicircles (lacking functional p53-specific binding sequence) with a size similar to that of the loop within the hcDNA, indicating that the extreme affinity of p53 for hcDNA is likely due to the binding of the protein to the hemicatenane. Although binding of p53 to hcDNA occurs in the absence of the nonspecific DNA-binding extreme carboxy-terminal regulatory domain (30-C, residues 363-393), the isolated 30-C domain (but not the sequence-specific p53 "core domain", residues 94-312) can also bind hcDNA. Only the full-length p53 can form stable ternary complexes with hcDNA and HMGB1. The possible biological relevance of p53 and HMGB1 binding to hemicatenanes is discussed.

Visualization of hemiknot DNA structure with an atomic force microscope.

The hemiknot, a novel type of DNA structure in which a loop is stabilized by threading one end of the duplex through another, has been studied in this paper. The hemiknot was obtained by reassociation of a DNA fragment with (CA/TG)n inserts of different lengths. Slow and fast migrating products were purified by gel electrophoresis and imaged by atomic force microscopy (AFM) using the aminopropylsilatrane-mica technique for sample preparation. Slow migrating product was characterized by the formation of small blobs for the short insert (60 bp) and clear loops and other morphologies for the long insert (188 bp). These structural features were found in almost 100% of the molecules of the slow migrating sample and were not present in the control sample. Measurements showed that the location of the blobs coincided with the positions of the inserts. The sample with the 188 bp insert in the 573 bp fragment had large structural irregularities. The majority of the molecules (77%) had asymmetrically located loops. The location of the loop in the molecules correlated well with the position of the insert in the fragment. The measured sizes of the loops were in agreement with the insert size. Altogether, these data support the hypothesis for hemiknot formation suggested earlier. In addition to looped structures, other morphologies of the hemiknot were identified in AFM images. Possible models for hemiknot formation and structure are discussed.

Structural analysis of hemicatenated DNA loops.

BACKGROUND: We have previously isolated a stable alternative DNA structure, which was formed in vitro by reassociation of the strands of DNA fragments containing a 62 bp tract of the CA-microsatellite poly(CA).poly(TG). In the model which was proposed for this structure the double helix is folded into a loop, the base of the loop consists of a DNA junction in which one of the strands of one duplex passes between the two strands of the other duplex, forming a DNA hemicatenane in a hemiknot structure. The hemiknot DNA structures obtained with long CA/TG inserts have been imaged by AFM allowing us to directly visualize the loops. RESULTS: Here we have analyzed this structure with several different techniques: high-resolution gel electrophoresis, probing by digestion with single stranded DNA-specific nucleases or with DNase I, modification with chemicals specific for unpaired bases, and atomic force microscopy. The data show a change in DNA structure localized to the CA/TG sequence and allow us to better understand the structure of this alternative conformation and the mechanism of its formation. CONCLUSIONS: The present work is in good agreement with the model of hemicatenated DNA loop proposed previously. In the presence of protein HMGB1, shifted reassociation of the strands of DNA fragments containing a tract of the poly(CA).poly(TG) microsatellite leads to the formation of DNA loops maintained at their base by a hemicatenated junction located within the repetitive sequence. No mobility of the junction along the DNA molecule could be detected under the conditions used. The novel possibility to prepare DNA hemicatenanes should be useful to further study this alternative DNA structure and its involvement in replication or recombination.