Václav Brázda

Cruciform structures are a common DNA feature important for regulating biological processes

DNA cruciforms play an important role in the regulation of natural processes involving DNA. These structures are formed by inverted repeats, and their stability is enhanced by DNA supercoiling. Cruciform structures are fundamentally important for a wide range of biological processes, including replication, regulation of gene expression, nucleosome structure and recombination. They also have been implicated in the evolution and development of diseases including cancer, Werners syndrome and others.Cruciform structures are targets for many architectural and regulatory proteins, such as histones H1 and H5, topoisomerase IIβ, HMG proteins, HU, p53, the proto-oncogene protein DEK and others. A number of DNA-binding proteins, such as the HMGB-box family members, Rad54, BRCA1 protein, as well as PARP-1 polymerase, possess weak sequence specific DNA binding yet bind preferentially to cruciform structures. Some of these proteins are, in fact, capable of inducing the formation of cruciform structures upon DNA binding. In this article, we review the protein families that are involved in interacting with and regulating cruciform structures, including (a) the junction-resolving enzymes, (b) DNA repair proteins and transcription factors, (c) proteins involved in replication and (d) chromatin-associated proteins. The prevalence of cruciform structures and their roles in protein interactions, epigenetic regulation and the maintenance of cell homeostasis are also discussed.

Tumor suppressor protein p53 binds preferentially to supercoiled DNA

Wild type human tumor suppressor protein p53 (expressed in insect cells) binds strongly to negatively supercoiled (sc) plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of scDNA in agarose gels and imaging by scanning force microscopy (SFM). The binding occurs both in the presence and absence of the p53 consensus sequence. At relatively low p53/DNA ratios, binding of p53 to scDNA results in the appearance of several retarded DNA bands on the gels, similar to a conventional topoisomer ladder generated enzymatically. However, after removal of p53 by deproteination, the original mobility of the scDNA is recovered, indicating that the reduction of torsional stress accompanying p53 binding does not reflect changes in linking number. In DNA samples partially relaxed by topoisomerase I p53 binds preferentially to the scDNA molecules with the largest negative superhelix density. SFM imaging of the p53/scDNA complex reveals a partial or total relaxation of the compact scDNA, the degree of which increases with the number of bound p53 molecules. Competition assays with linear DNA reveal a preference of p53 for scDNA. In addition, scDNA induces dissociation of p53 from a preformed complex with a DNA fragment (474 bp) containing the consensus sequence. We conclude that the affinity of p53 for negatively supercoiled DNA is greater than that for the consensus sequence in linear fragments. However, thermally denatured linearized plasmid DNA is efficient in competing for the binding of p53 to scDNA, although the first retarded band (presumed to contain one bound p53 molecule) is retained in the case of the plasmid containing the consensus sequence. Thus, it appears that interactions involving both the core domain and the C-terminal domain regulate the binding of p53 to scDNA. The above results are not restricted to human p53; the wild type rat p53 protein also results in the retardation of scDNA on agarose gels. The biological implications of the novel DNA binding activities of p53 are discussed.

Effect of transition metals on binding of p53 protein to supercoiled DNA and to consensus sequence in DNA fragments

Recently we have shown that wild-type human p53 protein binds preferentially to supercoiled (sc) DNA in vitro in both the presence and absence of the p53 consensus sequence (p53CON). This binding produces a ladder of retarded bands on an agarose gel. Using immunoblotting with the antibody DO-1, we show that the bands obtained correspond to ethidium-stained DNA, suggesting that each band of the ladder contains a DNA-p53 complex. The intensity and the number of these bands are decreased by physiological concentrations of zinc ions. At higher zinc concentrations, binding of p53 to scDNA is completely inhibited. The binding of additional zinc ions to p53 appears much weaker than the binding of the intrinsic zinc ion in the DNA binding site of the core domain. In contrast to previously published data suggesting that 100 μM zinc ions do not influence p53 binding to p53CON in a DNA oligonucleotide, we show that 5 – 20 μM zinc efficiently inhibits binding of p53 to p53CON in DNA fragments. We also show that relatively low concentrations of dithiothreitol but not of 2-mercaptoethanol decrease the concentration of free zinc ions, thereby preventing their inhibitory effect on binding of p53 to DNA. Nickel and cobalt ions inhibit binding of p53 to scDNA and to its consensus sequence in linear DNA fragments less efficiently than zinc; cobalt ions are least efficient, requiring >100 μM Co2+ for full inhibition of p53 binding. Modulation of binding of p53 to DNA by physiological concentrations of zinc might represent a novel pathway that regulates p53 activity in vivo.

Satellite glial cells express IL-6 and corresponding signal-transducing receptors in the dorsal root ganglia of rat neuropathic pain model.

There is a growing body of evidence that cytokines contribute to both induction and maintenance of neuropathic pain derived from changes in dorsal root ganglia (DRG), including the activity of the primary sensory neurons and their satellite glial cells (SGC). We used immunofluorescence and in situ hybridization methods to provide evidence that chronic constriction injury (CCI) of the sciatic nerve induces synthesis of interleukin-6 (IL-6) in SGC, elevation of IL-6 receptor (IL-6R) and activation of signal transducer and activator of transcription 3 (STAT3) signalling. Unilateral CCI of the rat sciatic nerve induced mechanoallodynia and thermal hyperalgesia in ipsilateral hind paws, but contralateral paws exhibited only temporal changes of sensitivity. We demonstrated that IL-6 mRNA and protein, which were expressed at very low levels in naïve DRG, were bilaterally increased not only in L4-L5 DRG neurons but also in SGC activated by unilateral CCI. Besides IL-6, substantial increase of IL-6R and pSTAT3 expression occurred in SGC following CCI, however, IL-6R associated protein, gp130 levels did not change. The results may suggest that unilateral CCI of the sciatic nerve induces bilateral activation of SGC in L4-L5 DRG to transduce IL-6 signalling during neuroinflammation.

New ELISA technique for analysis of p53 protein/DNA binding properties

The p53 tumour suppressor protein is one of the most important topics in cancer research. Its function is associated with the ability to bind DNA in a sequence-specific manner and to operate as a transcription factor. In the present study, we have developed a rapid and reliable method for analysing sequence-specific binding of p53 protein to DNA using a modified enzyme-linked immunosorbent assay (ELISA). In this p53/DNA-ELISA, we use streptavidin-coated microplates to capture biotinylated oligonucleotides containing p53 consensus sequences (p53CON). This newly developed nonradioactive assay allows the detection of p53/DNA complexes using different monoclonal antibodies recognising p53 and has comparable or higher sensitivity to more complicated radioactive methods. Using this method, we can detect binding of endogenous p53 to p53CON and activation of p53 protein for sequence-specific DNA binding. Variations of the basic protocol have also been developed to perform competition experiments and to study p53 binding to natural binding sequences. This modified DNA-ELISA is applicable for screening p53 binding properties from various sources in a short time.

Spatio-temporal changes of SDF1 and its CXCR4 receptor in the dorsal root ganglia following unilateral sciatic nerve injury as a model of neuropathic pain

There is a growing evidence that chemokines and their receptors play a role in inducing and maintaining neuropathic pain. In the present study, unilateral chronic constriction injury (CCI) of rat sciatic nerve under aseptic conditions was used to investigate changes for stromal derived factor-1 (SDF1) and its CXCR4 receptor in lumbal (L4–L5) and cervical (C7–C8) dorsal root ganglia (DRG) from both sides of naïve, CCI-operated and sham-operated rats. All CCI-operated rats displayed mechanical allodynia and thermal hyperalgesia in hind paws ipsilateral to CCI, but forepaws exhibited only temporal changes of sensitivity not correlated with alterations in SDF1 and CXCR4 proteins. Naïve DRG displayed immunofluorescence for SDF1 (SDF1-IF) in the satellite glial cells (SGC) and CXCR4-IF in the neuronal bodies with highest intensity in small- and medium-sized neurons. Immunofluorescence staining and Western blot analysis confirmed that unilateral CCI induced bilateral alterations of SDF1 and CXCR4 proteins in both L4–L5 and C7–C8 DRG. Only lumbal DRG were invaded by ED-1+ macrophages exhibiting SDF1-IF while elevation of CXCR4-IF was found in DRG neurons and SGC but not in ED-1+ macrophages. No attenuation of mechanical allodynia, but reversed thermal hyperalgesia, in ipsi- and contralateral hind paws was found in CCI-operated rats after i.p. administration of CXCR4 antagonist (AMD3100). These results indicate that SDF1/CXCR4 changes are not limited to DRG associated with injured nerve but that they also spread to DRG non-associated with such nerve. Functional involvement of these alterations in DRG non-associated with injured nerve in neuropathic pain remains to be elucidated.

Interferon-Inducible Protein 16: Insight into the Interaction with Tumor Suppressor p53

IFI16 is a member of the interferon-inducible HIN-200 family of nuclear proteins. It has been implicated in transcriptional regulation by modulating protein-protein interactions with p53 tumor suppressor protein and other transcription factors. However, the mechanisms of interaction remain unknown. Here, we report the crystal structures of both HIN-A and HIN-B domains of IFI16 determined at 2.0 and 2.35 Å resolution, respectively. Each HIN domain comprises a pair of tightly packed OB-fold subdomains that appear to act as a single unit. We show that both HIN domains of IFI16 are capable of enhancing p53-DNA complex formation and transcriptional activation via distinctive means. HIN-A domain binds to the basic C terminus of p53, whereas the HIN-B domain binds to the core DNA-binding region of p53. Both interactions are compatible with the DNA-bound state of p53 and together contribute to the effect of full-length IFI16 on p53-DNA complex formation and transcriptional activation.

Enhancement of p53 sequence-specific binding by DNA supercoiling

Using a new competition assay, we investigated the effect of DNA negative supercoiling on the DNA sequence-specific binding (SSDB) of human wild-type (wt) p53 protein. We found that supercoiled (sc) pBluescript DNAs with different inserted p53 target sequences were stronger competitors than a mixture of scDNA pBluescript with the given 20-mer target oligodeoxynucleotide. ScDNAs were always better competitors than their linearized or relaxed forms. Two DNAs with extruded cruciforms within the target sequence were the best competitors; removal of the cruciforms resulted in a decrease of competitor strength. In contrast to the full-length wt p53, the deletion mutant p53CΔ30 and the p53 core domain (93–312 aa) showed no enhancement of p53 SSDB to scDNA, suggesting that, in addition to the p53 core domain, the C-terminal was involved in this binding. We conclude that cruciforms and DNA bends contribute to the enhancement of p53 SSDB to scDNA and that the DNA supercoiling is an important determinant in the p53 sequence-specific binding. Supercoiling may thus play a significant role in the complex p53-regulatory network.

Preferential binding of IFI16 protein to cruciform structure and superhelical DNA.

Interferon (IFN)-inducible HIN-200 proteins play an important role in transcriptional regulation linked to cell cycle control, inflammation, autoimmunity and differentiation. IFI16 has been identified as a target of IFNα and γ and is a member of the HIN-200 protein family. Expression level of IFI16 is often decreased in breast cancers, implicating its role as a tumor suppressor. As a potent transcription factor, IFI16 possesses a transcriptional regulatory region, a PYD/DAPIN/PAAD region which associates with IFN response, DNA-binding domains and binding regions for tumor suppressor proteins BRCA1 and p53. It is also reported that IFI16 protein is capable of binding p53 and cMYC gene promoters. Here, we demonstrate that IFI16 protein binds strongly to negatively superhelical plasmid DNA at a native superhelix density, as evidenced by electrophoretic retardation of supercoiled (sc) DNA in agarose gels. Binding of IFI16 to supercoiled DNA results in the appearance of one or more retarded DNA bands on the gels. After removal of IFI16, the original mobility of the scDNA is recovered. By contrast, IFI16 protein binds very weakly to the same DNA in linear state. Using short oligonucleotide targets, we also detect a strong preference for IFI16 binding to cruciform DNA structure compared to linear DNA topology. Hence, this novel DNA-binding property of IFI16 protein to scDNA and cruciform structures may play critical roles in its tumor suppressor function.

Bilateral elevation of interleukin-6 protein and mRNA in both lumbar and cervical dorsal root ganglia following unilateral chronic compression injury of the sciatic nerve

BackgroundCurrent research implicates interleukin (IL)-6 as a key component of the nervous-system response to injury with various effects.MethodsWe used unilateral chronic constriction injury (CCI) of rat sciatic nerve as a model for neuropathic pain. Immunofluorescence, ELISA, western blotting and in situ hybridization were used to investigate bilateral changes in IL-6 protein and mRNA in both lumbar (L4-L5) and cervical (C7-C8) dorsal root ganglia (DRG) following CCI. The operated (CCI) and sham-operated (sham) rats were assessed after 1, 3, 7, and 14 days. Withdrawal thresholds for mechanical hyperalgesia and latencies for thermal hyperalgesia were measured in both ipsilateral and contralateral hind and fore paws.ResultsThe ipsilateral hind paws of all CCI rats displayed a decreased threshold of mechanical hyperalgesia and withdrawal latency of thermal hyperalgesia, while the contralateral hind and fore paws of both sides exhibited no significant changes in mechanical or thermal sensitivity. No significant behavioral changes were found in the hind and fore paws on either side of the sham rats, except for thermal hypersensitivity, which was present bilaterally at 3 days. Unilateral CCI of the sciatic nerve induced a bilateral increase in IL-6 immunostaining in the neuronal bodies and satellite glial cells (SGC) surrounding neurons of both lumbar and cervical DRG, compared with those of naive control rats. This bilateral increase in IL-6 protein levels was confirmed by ELISA and western blotting. More intense staining for IL-6 mRNA was detected in lumbar and cervical DRG from both sides of rats following CCI. The DRG removed from sham rats displayed a similar pattern of staining for IL-6 protein and mRNA as found in naive DRG, but there was a higher staining intensity in SGC.ConclusionsBilateral elevation of IL-6 protein and mRNA is not limited to DRG homonymous to the injured nerve, but also extended to DRG that are heteronymous to the injured nerve. The results for IL-6 suggest that the neuroinflammatory reaction of DRG to nerve injury is propagated alongside the neuroaxis from the lumbar to the remote cervical segments. This is probably related to conditioning of cervical DRG neurons to injury.