Atomic force microscopy shows that vaccinia topoisomerase IB generates filaments on DNA in a cooperative fashion

Type IB DNA topoisomerases cleave and rejoin one strand of the DNA duplex, allowing for the removal of supercoils generated during replication and transcription. In addition, electron microscopy of cellular and viral TopIB–DNA complexes has suggested that the enzyme promotes long-range DNA–DNA crossovers and synapses. Here, we have used the atomic force microscope to visualize and quantify the interaction between vaccinia topoisomerase IB (vTopIB) and DNA. vTopIB was found to form filaments on nicked-circular DNA by intramolecular synapsis of two segments of a single DNA molecule. Measuring the filament length as a function of protein concentration showed that synapsis is a highly cooperative process. At high protein:DNA ratios, synapses between distinct DNA molecules were observed, which led to the formation of large vTopIB-induced DNA clusters. These clusters were observed in the presence of Mg(2+), Ca(2+) or Mn(2+), suggesting that the formation of intermolecular vTopIB-mediated DNA synapsis is favored by screening of the DNA charge

A 27 kDa protein binds to a positive and a negative regulatory sequence in the promoter of the ICL1 gene from Saccharomyces cerevisiae

6 pages, 6 figures, 1 table.IsocitrateICL1, is one of the key enzymes of the glyoxylate pathway, which operates as an anaplerotic route for replenishing the tricarboxylic acid cycle; it is required for growth of Saccharomyces cerevisiae on carbon sources such as ethanol, but is dispensable when fermentable carbon sources are available. The positive regulation of the ICL1 gene by an upstream activating sequence (UAS) element located between -397 and -388 has been previously reported. In this paper we show that the ICL1 promoter sequence 5'-AGTCCGGACTAGCATCCCAG-3' located between -261 and -242 contains an upstream repressing sequence (URS) element. We have identified and partially purified a 27 kDa protein that binds specifically to both the UAS and URS sequences of the ICL1 promoter. For both UAS and URS, binding requires the protein Snf1 (Cat1), a protein kinase essential for the derepression of genes repressed by glucose. Binding does not take place with extracts from glucose-grown strains, unless they lack Mig1, a negative regulatory protein involved in glucose repression.This work was supported by grants PB94-0091-C02-01 and PB94-0091-C02-02 from the DGICYT.Peer reviewe

Structural analysis of hyperperiodic DNA from Caenorhabditis elegans

Several bioinformatics studies have identified an unexpected but remarkably prevalent ∼10 bp periodicity of AA/TT dinucleotides (hyperperiodicity) in certain regions of the Caenorhabditis elegans genome. Although the relevant C.elegans DNA segments share certain sequence characteristics with bent DNAs from other sources (e.g. trypanosome mitochondria), the nematode sequences exhibit a much more extensive and defined hyperperiodicity. Given the presence of hyperperiodic structures in a number of critical C.elegans genes, the physical characteristics of hyperperiodic DNA are of considerable interest. In this work, we demonstrate that several hyperperiodic DNA segments from C.elegans exhibit structural anomalies using high-resolution atomic force microscopy (AFM) and gel electrophoresis. Our quantitative analysis of AFM images reveals that hyperperiodic DNA adopts a significantly smaller mean square end-to-end distance, hence a more compact coil structure, compared with non-periodic DNA of similar length. While molecules remain capable of adopting both bent and straight (rod-like) configurations, indicating that their flexibility is still retained, examination of the local curvatures along the DNA contour length reveals that the decreased mean square end-to-end distance can be attributed to the presence of long-scale intrinsic bending in hyperperiodic DNA. Such bending is not detected in non-periodic DNA. Similar studies of shorter, nucleosome-length DNAs that survived micrococcal nuclease digestion show that sequence hyperperiodicity in short segments can likewise induce strong intrinsic bending. It appears, therefore, that regions of the C.elegans genome display a significant correlation between DNA sequence and unusual mechanical properties

Bulk and single-molecule analysis of a bacterial DNA2-like helicase-nuclease reveals a single-stranded DNA looping motor

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.Wellcome Trust [100401/Z/12/Z to M.D.]; EuropeanResearch Council [681299 to F.M.-H.]; Spanish Min-istry of Economy and Competitiveness [BFU2017-83794-PAEI/FEDER, UE to F.M.-H.]; Comunidad de MadridTec4Bio [S2018/NMT-4443 to F.M.-H.]; NanoBioCancer[Y2018/BIO-4747 to F.M.-H.]. Funding for open accesscharge: Wellcome Trust [100401/Z/12/Z].Peer reviewe

Determination of a lectin protein allergen in food by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An aptamer affinity sorbent was prepared for clean-up, preconcentration, separation and characterization of a food allergen protein by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS). SPE microcartridges were packed with a sorbent based on magnetic bead particles modified with an aptamer against the target lectin protein concanavalin A (Con A). After optimization of several parameters of the SPE-CE-MS method, the sample (ca. 30 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while the retained protein was eluted with 100 mM NH4OH (pH 11.2) (ca. 100 nL). The developed method was linear between 0.5 and 20 mg·L-1 and the limit of detection (LOD) was 0.25 mg·L-1, which was 100 times lower than by CE-MS. The repeatability of the method was satisfactory, with relative standard deviations (RSD) for migration times and peak areas below 1.9 and 8.1%, respectively. In addition, the microcartridges could be reused more than 25 analyses without significant loss of extraction efficiency. Finally, the applicability and versatility of the developed method were demonstrated by analyzing low levels of Con A in different food matrices (i.e. white beans, as well as chickpea, lentils, and wheat flours), leading to satisfactory results, with recoveries between 87 and 115%

Accurate determination of the milk protein allergen β-lactoglobulin by on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry

An on-line aptamer affinity solid-phase extraction capillary electrophoresis-mass spectrometry (AA-SPE-CE-MS) method was developed to purify, preconcentrate, separate, and characterize the milk allergenic protein β-lactoglobulin (β-LG) in food samples. The sorbent to pack into the SPE microcartidges was prepared by immobilizing an aptamer against β-LG onto magnetic bead particles. After optimizing the SPE-CE-MS method, the sample (ca. 75 µL) was loaded in separation background electrolyte (BGE, 2 M acetic acid pH 2.2), while a solution of 100 mM NH4OH (pH 11.2) (ca. 100 nL) was used for the protein elution. The linearity of the method ranged between 0.1 and 20 µg·mL-1 and the limit of detection (LOD) was 0.05 µg·mL-1, which was 200 times lower than by CE-MS. The method was repeatable in terms of relative standard deviation (RSD) for migration times and peak areas (< 0.5 % and 2.4 %, respectively) and microcartridge lifetime was more than 25 analyses. The applicability of the method for the determination of low levels of β-LG was shown by analyzing milk-free foods (i.e. a 100 % cocoa dark chocolate, a hypoallergenic formula for infants, and a dairy-free white bread) and milk-containing white breads. Results were satisfactory in all cases, thus demonstrating the great potential of the developed method for accurate food safety and quality control