Protein Content of Circulating Nucleoprotein Complexes.

In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples. Fourteen proteins previously described as tumor specific proteins were found in cancer patients whereas not one of them was found in healthy individuals. The data obtained demonstrate the involvement of different cellular and extracellular proteins in circulating cell-free DNA.

Reciprocal Self-Assembly of Peptide-DNA Conjugates into a Programmable Sub-10-nm Supramolecular Deoxyribonucleoprotein.

To overcome the limitations of molecular assemblies, the development of novel supramolecular building blocks and self-assembly modes is essential to create more sophisticated, complex, and controllable aggregates. The self-assembly of peptide-DNA conjugates (PDCs), in which two orthogonal self-assembly modes, that is, ß-sheet formation and Watson-Crick base pairing, are covalently combined in one supramolecular system, is reported. Despite extensive research, most self-assembly studies have focused on using only one type of building block, which restricts structural and functional diversity compared to multicomponent systems. Multicomponent systems, however, suffer from poor control of self-assembly behaviors. Covalently conjugated PDC building blocks are shown to assemble into well-defined and controllable nanostructures. This controllability likely results from the decrease in entropy associated with the restriction of the molecular degrees of freedom by the covalent constraints. Using this strategy, the possibility to thermodynamically program nano-assemblies to exert gene regulation activity with low cytotoxicity is demonstrated.

An archaeal RadA paralog influences presynaptic filament formation.

Recombinases of the RecA family play vital roles in homologous recombination, a high-fidelity mechanism to repair DNA double-stranded breaks. These proteins catalyze strand invasion and exchange after forming dynamic nucleoprotein filaments on ssDNA. Increasing evidence suggests that stabilization of these dynamic filaments is a highly conserved function across diverse species. Here, we analyze the presynaptic filament formation and DNA binding characteristics of the Sulfolobus solfataricus recombinase SsoRadA in conjunction with the SsoRadA paralog SsoRal1. In addition to constraining SsoRadA ssDNA-dependent ATPase activity, the paralog also enhances SsoRadA ssDNA binding, effectively influencing activities necessary for presynaptic filament formation. These activities result in enhanced SsoRadA-mediated strand invasion in the presence of SsoRal1 and suggest a filament stabilization function for the SsoRal1 protein.

One-off streptococcal serologic testing in young children with recurrent tonsillitis.

OBJECTIVES: Recurrent acute tonsillitis in children under 4 years of age is usually viral, making antibiotic therapy inappropriate and the indication for tonsillectomy uncertain. Identifying those young children with bacterial infections is therefore important. The purpose of this study was to determine whether one-off streptococcal serologic testing is a useful tool in assessing recurrent acute tonsillitis in young children. METHODS: We performed a retrospective study of 45 children (35 male and 10 female) under the age of 4 years who were found by a staff otolaryngologist to have recurrent acute tonsillitis over a 5-year period and had one-off serologic testing for anti-streptolysin O titers and anti-deoxyribonuclease B levels. Data were collected by chart review. RESULTS: Three children (6.7%) had clearly positive titers for either one or both streptococcal antibodies. Children with negative serologic results were significantly less likely to have shown a significant response to antibiotic therapy for their acute episodes (26% versus 100%; p = .026). Nine children (20%) eventually underwent tonsillectomy, all of whom had negative serologic results. CONCLUSIONS: Anti-streptolysin O and anti-deoxyribonuclease B levels may aid clinical evaluation of recurrent acute tonsillitis in young children in differentiating between those cases due to group A beta-hemolytic Streptococcus and those that are viral in origin.

Separation of nearly identical repeats in shotgun assemblies using defined nucleotide positions, DNPs.

An increasingly important problem in genome sequencing is the failure of the commonly used shotgun assembly programs to correctly assemble repetitive sequences. The assembly of non-repetitive regions or regions containing repeats considerably shorter than the average read length is in practice easy to solve, while longer repeats have been a difficult problem. We here present a statistical method to separate arbitrarily long, almost identical repeats, which makes it possible to correctly assemble complex repetitive sequence regions. The differences between repeat units may be as low as 1% and the sequencing error may be up to ten times higher. The method is based on the realization that a comparison of only a part of all overlapping sequences at a time in a data set does not generate enough information for a conclusive analysis. Our method uses optimal multi-alignments consisting of all the overlaps of each read. This makes it possible to determine defined nucleotide positions, DNPs, which constitute the differences between the repeat units. Differences between repeats are distinguished from sequencing errors using statistical methods, where the probabilities of obtaining certain combinations of candidate DNPs are calculated using the information from the multi-alignments. The use of DNPs and combinations of DNPs will allow for optimal and rapid assemblies of repeated regions. This method can solve repeats that differ in only two positions in a read length, which is the theoretical limit for repeat separation. We predict that this method will be highly useful in shotgun sequencing in the future.

Gel-based fluorescence resonance energy transfer (gelFRET) analysis of nucleoprotein complex architecture.

A gel-based fluorescence resonance energy transfer (gelFRET) assay was developed for analysis of the architecture of nucleoprotein complexes. gelFRET is based on fluorescence analysis of nucleoprotein complexes separated by polyacrylamide gel electrophoresis. These complexes are separated from free components and nonspecific complexes, enabling fluorescence analysis of complexes containing all components in stoichiometric proportions. gelFRET can be used to investigate the structural organization of nucleoprotein complexes through comparison of the relative efficiencies of energy transfer from donor fluorophores linked to different positions on DNA to an acceptor fluorophore linked to a unique position on the binding protein. We have applied gelFRET to analysis of the orientation of binding by heterodimeric transcription factors. By using Fos-Jun heterodimers as a model system we have identified the structural determinants that control the orientation of heterodimer binding. gelFRET can be applied to studies of a variety of biological processes that influence the proximity of two sites within a complex, such as the assembly of transcription regulatory complexes.

A novel assay for examining the molecular reactions at the eukaryotic replication fork: activities of replication protein A required during elongation.

Studies to elucidate the reactions that occur at the eukaryotic replication fork have been limited by the model systems available. We have established a method for isolating and characterizing Simian Virus 40 (SV40) replication complexes. SV40 rolling circle complexes are isolated using paramagnetic beads and then incubated under replication conditions to obtain continued elongation. In rolling circle replication, the normal mechanism for termination of SV40 replication does not occur and the elongation phase of replication is prolonged. Thus, using this assay system, elongation phase reactions can be examined in the absence of initiation or termination. We show that the protein requirements for elongation of SV40 rolling circles are equivalent to complete SV40 replication reactions. The DNA produced by SV40 rolling circles is double-stranded, unmethylated and with a much longer length than the template DNA. These properties are similar to those of physiological replication forks. We show that proteins associated with the isolated rolling circles, including SV40 T antigen, DNA polymerase alpha, replication protein A (RPA) and RF-C, are necessary for continued DNA synthesis. PCNA is also required but is not associated with the isolated complexes. We present evidence suggesting that synthesis of the leading and lagging strands are co-ordinated in SV40 rolling circle replication. We have used this system to show that both RPA-protein and RPA-DNA interactions are important for RPA's function in elongation.

Nucleoprotein gene tracking: localization of specific HIV-1 genes to subchromatin nucleoprotein complexes containing endonuclease activity in HIV-1-infected human cells.

We developed a technique with which to isolate specific subchromatin deoxyribonucleoprotein/ribonucleoprotein precursor complexes containing discrete genes from intact mammalian nuclei by direct restriction enzyme treatment with MspI. These nucleoprotein complexes can be further fractionated and purified by two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electroelution and removal of detergent, virtually thousands of nucleoprotein complexes can be examined for the presence of tightly bound genes and enzymatic activities. Nucleoprotein gene tracking procedures were applied to study the acidic nucleoprotein complexes from steady-state human H9 cells uninfected or infected with human immunodeficiency virus type 1 (HIV-1) virus. The purified nucleoprotein complexes were screened for the presence of loosely and tightly associated HIV-1 gene sequences (pol, env, tat, and rev) using a DNA hybridization protocol. In HIV-1-infected cells, four specific nucleoprotein complexes out of several hundred were found to contain tightly bound HIV-1 pol gene sequences after purification. By contrast, the other HIV-1 gene sequences (env, tat, and rev) were not tightly bound to any of the nucleoprotein complexes in HIV-infected cells. The observations suggest that certain HIV-1 genes associate with specific chromatin nucleoprotein complexes, regardless of their pattern of DNA integration into the human genome. At least two of the HIV-1 pol-containing nucleoprotein complexes of apparent M(r) approximately 94,000, pI approximately 6.5, and M(r) approximately 47,000, pI approximately 5.1 contain DNA endonuclease activity. This was confirmed in the present study, using linearized pUC19 plasmid substrate. The technique can be used to study a variety of problems concerning the association of specific genes and enzymes with specific nucleoprotein complexes J. Cell. Biochem. Suppls. 32/33:158-165, 1999.

Perturbation of nucleosome core structure by the SWI/SNF complex persists after its detachment, enhancing subsequent transcription factor binding.

To investigate the mechanism of SWI/SNF action, we have analyzed the pathway by which SWI/SNF stimulates formation of transcription factor-bound nucleosome core complexes. We report here that the SWI/SNF complex binds directly to nucleosome cores and uses the energy of ATP hydrolysis to disrupt histone/DNA interactions, altering the preferred path of DNA bending around the histone octamer. This disruption occurs without dissociating the DNA from the surface of the histone octamer. ATP-dependent disruption of nucleosomal DNA by SWI/SNF generates an altered nucleosome core conformation that can persist for an extended period after detachment of the SWI/SNF complex. This disrupted conformation retains an enhanced affinity for the transcription factor GAL4-AH. Thus, ATP-dependent nucleosome core disruption and enhanced binding of the transcription factor can be temporally separated. These results indicate that SWI/SNF can act transiently in the remodeling of chromatin structure, even before interactions of transcription factors.

Reactive arthritis associated with group C and group G beta-hemolytic streptococci.

OBJECTIVE: Group A beta-hemolytic streptococci (GAS) are known to be capable of evoking sterile arthritis. Reactive arthritis (ReA) has been reported sporadically following primary infection with group C and group G beta-hemolytic streptococci (GCS, GGS). We prospectively studied 4 cases of ReA secondary to throat infection with GCS and GGS. METHODS: Four patients with arthritis secondary to throat infection were seen. Three patients were Dutch, one was Indonesian; female/male ratio was 1/3; mean age was 30 years (range 18-46). Diagnostic evaluation included culture of throat swab and serological screening. RESULTS: All patients presented with a nonmigratory asymmetrical arthritis: monoarthritis in one patient, oligoarthritis in 3. Culture of throat swab was positive in all. Antistreptolysin-O (ASO) titer rose significantly in 2 patients, and anti-DNase-B rose in 2 patients. ASO was maximal (mean 1000 U/ml; range 890-1110) and anti-DNase-B was 395 U/ml (range 290-500). Treatment consisted of feneticillin for 5 days; nonsteroidal antiinflammatory drugs were prescribed on demand. All patients recovered fully in 3 to 12 weeks. CONCLUSION: These cases provide evidence of a benign non-group A streptococcal ReA, i.e., secondary to GCS or GGS. The presence of the organism in the throat along with the elevation of antibody to streptococcal products is important for the diagnosis of GCS/GGS associated ReA. A positive throat culture is needed for differentiation from GAS associated poststreptococcal ReA, because prophylactic measures are effective only in GAS associated sequelae, but not in GCS/GGS associated ReA.

Alterations in chromosome structure and variations in the inherent radiation sensitivity of human cells.

Variations in the inherent radiosensitivity of both tumor cells and the normal tissues that surround them play an important role in tumor response to radiation therapy. In vitro studies suggest that variations in radiation sensitivity both between different tissues and within a specific histology are a reflection of differences in the rate and fidelity of rejoining of chromosome breaks. Cells of radiosensitive cell lines rejoin breaks more slowly and with less fidelity than those of more resistant cell lines. Differences in radiation sensitivity are also associated with variations in chromosome structure as detected by nucleoid-based assays. A model is presented to suggest that the radiation sensitivity of a cell line is a reflection of its transcriptional architecture, the number and genomic location of its actively transcribing regions. Also, it is proposed that chromosome breaks induced at or near transcriptionally active regions of the genome are rejoined preferentially and with greater fidelity than breaks induced at other regions of the genome.

DNA-binding domains of Fos and Jun do not induce DNA curvature: an investigation with solution and gel methods.

We demonstrate the use of a DNA minicircle competition binding assay, together with DNA cyclization kinetics and gel-phasing methods, to show that the DNA-binding domains (dbd) of the heterodimeric leucine zipper protein Fos-Jun do not bend the AP-1 target site. Our DNA constructs contain an AP-1 site phased by 1-4 helical turns against an A-tract-directed bend. Competition binding experiments reveal that (dbd)Fos-Jun has a slight preference for binding to linear over circular AP-1 DNAs, independent of whether the site faces in or out on the circle. This result suggests that (dbd)Fos-Jun slightly stiffens rather than bends its DNA target site. A single A-tract bend replacing the AP-1 site is readily detected by its effect on cyclization kinetics, in contrast to the observations for Fos-Jun bound at the AP-1 locus. In contrast, comparative electrophoresis reveals that Fos-Jun-DNA complexes, in which the A-tract bend is positioned close (1-2 helical turns) to the AP-1 site, show phase-dependent variations in gel mobilities that are comparable with those observed when a single A-tract bend replaces the AP-1 site. Whereas gel mobility variations of Fos-Jun-DNA complexes decrease linearly with increasing Mg2+ contained in the gel, the solution binding preference of (dbd)Fos-Jun for linear over circular DNAs is independent of Mg2+ concentration. Hence, gel mobility variations of Fos-Jun-DNA complexes are not indicative of (dbd)Fos-Jun-induced DNA bending (upper limit 5 degrees) in the low salt conditions of gel electrophoresis. Instead, we propose that the gel anomalies depend on the steric relationship of the leucine zipper region with respect to a DNA bend.

Characterization of the ATF/CREB site and its complex with GCN4.

We have studied DNA minicircles containing the ATF/CREB binding site for GCN4 by using a combination of cyclization kinetics experiments and Monte Carlo simulations. Cyclization rates were determined with and without GCN4 for DNA constructs containing the ATF/CREB site separated from a phased A-tract multimer bend by a variable length phasing adaptor. The cyclization results show that GCN4 binding does not significantly change the conformation of the ATF/CREB site, which is intrinsically slightly bent toward the major groove. Monte Carlo simulations quantitate the ATF/CREB site structure as an 8 degrees bend toward the major groove in a coordinate frame near the center of the site. The ATF/CREB site is underwound by 53 degrees relative to the related AP-1 site DNA. The effect of GCN4 binding can be modeled either as a decrease in the local flexibility, corresponding to an estimated 60% increase in the persistence length for the 10-bp binding site, or possibly as a small decrease (1 degrees) in intrinsic bend angle. Our results agree with recent electrophoretic and crystallographic studies and demonstrate that cyclization and simulation can characterize subtle changes in DNA structure and flexibility.

A previously unidentified host protein protects retroviral DNA from autointegration.

Integration of a DNA copy of the viral genome into a host chromosome is an essential step in the retrovirus life cycle. The machinery that carries out the integration reaction is a nucleoprotein complex derived from the core of the infecting virion. To successfully integrate into host DNA, the viral DNA within this complex must avoid self-destructive integration into itself, a reaction termed autointegration. We have previously shown [Lee, M. S. and Craigie, R. (1994) Proc. Natl. Acad. Sci. USA 91, 9823-9827] that viral nucleoprotein complexes isolated from Moloney murine leukemia virus-infected cells exhibit a barrier to autointegration. This autointegration barrier could be destroyed by stripping factors from the complexes and subsequently restored by incubation with a host cell extract, but not by incubation with an extract of disrupted virions. We have now used this autointegration barrier reconstitution assay to purify the host factor from uninfected NIH 3T3 fibroblasts. It is a single polypeptide of 89 aa that does not match any previously identified protein. The identity of the protein was confirmed by expressing it in Escherichia coli and demonstrating the activity of the heterologously expressed protein in the reconstitution assay.

A novel DNA recognition mode by the NF-kappa B p65 homodimer.

The crystal structure of the NF-kappa B p65 (RelA) homodimer in complex with a DNA target has been determined to 2.4 A resolution. The two p65 subunits are not symmetrically disposed on the DNA target. The homodimer should optimally bind to a pseudo-palindromic nine base pair target with each subunit recognizing a 5'GGAA-3' half site separated by a central A-T base pair. However, one of the subunits (subunit B) encounters a half site of 5'-GAAA-3'. The single base-pair change from G-C to A-T results in highly unfavorable interactions between this half site and the base contacting protein residues in subunit B, which leads to an 18 degrees rotation of the N-terminal terminal domain from its normal conformation. Remarkably, subunit B retains all the interactions with the sugar phosphate backbone of the DNA target. This mode of interaction allows the NF-kappa B p65 homodimer to recognize DNA targets containing only one cognate half site. Differences in the sequence of the other half site provide variations in conformation and affinity of the complex.

Interaction of the Rb tumor suppressor protein with the c-fos promoter in c-fos transfected cells overexpressing c-fos and Rb.

The Rb tumor suppressor protein is overexpressed in HeLa cell lines permanently transfected with a constitutively expressed c-fos gene. CP17-14 cell overexpression of Rb may be due to a balancing response to overexpression of the stimulatory effects of c-fos overexpression on transcription. The cis-acting retinoblastoma control element (RCE, -97 to -86 bp) in the human c-fos promoter is thought to allow regulation of c-fos by Rb. Gel-shift assays were performed with a 168 bp fragment encoding the c-fos RCE. Competition assays with increasing mass of unlabeled probe or dose-dependence assays using increasing mass of nuclear proteins, demonstrated sequence-specific complex formation. Indistinguishable complexes were formed between the c-fos RCE fragment in transfected cells, but at higher levels (> 50%), compared to proteins from parental cells. Supershift analysis utilizing epitope-specific Rb-monoclonal antibodies indicated the presence of Rb protein bound to the RCE-containing DNA fragment. In contrast, polyclonal anti-Rb antibodies enhanced the amounts of nuclear protein-DNA complexes detected but did not result in a supershift. These results suggested the presence of Rb and/or Rb-like peptides involved in complex formation and the presence of multiple variants of RCE-binding complexes in response to c-fos over-expression.

Natural resistance of acute myeloid leukemia cell lines to mitoxantrone is associated with lack of apoptosis.

The purpose of this study was to characterize mitoxantrone-induced cytotoxicity in KG1a and TF-1, two P-glycoprotein expressing AML cell lines which display early differentiation phenotypes, compared to more mature HL-60 and U937 cells. KG1a and TF-1 cells were found to be 30-40-fold more resistant to mitoxantrone than HL-60 and U937 cells. Uptake and efflux of mitoxantrone were similar for all cell lines. Moreover, a potent P-glycoprotein blocker (PSC833) had no impact on either accumulation or efflux. No differences were found in the appearance and removal of mitoxantrone-induced DNA-protein complexes. These results suggest that resistance of KG1a and TF-1 cells is not related to a decreased interaction between mitoxantrone and topoisomerase II. Further studies showed that the mechanisms of cell death were different for sensitive and resistant cell lines. Thus, mitoxantrone induced rapid apoptotic cell death in sensitive cells as indicated by characteristic morphological changes and both high molecular weight and internucleosomal DNA fragmentation. In contrast, mitoxantrone induced a G2-M block in resistant cells followed by a progressive loss of viability with necrotic features. Neither oligonucleosomal nor large DNA fragments were detected in these cells during a post-treatment period of up to 96 h. Finally, drug-induced activation of the AP-1 transcription factor was higher in resistant cell lines than in sensitive ones whereas activation of NF-kappaB was comparable. Therefore, our study provides evidence that certain AML cells display natural resistance to mitoxantrone which is independent of drug transport and drug-target interactions but appears to be associated with the inability of the drug to induce apoptosis in these cells.