Low Concentrations Of MgCl2 Research Articles

Facile Fabrication of Natural Polyelectrolyte-Nanoclay Composites: Halloysite Nanotubes, Nucleotides and DNA Study

Complexation of biopolymers with halloysite nanotubes (HNTs) can greatly affect their applicability as materials building blocks. Here we have performed a systematic investigation of fabrication of halloysite nanotubes complexes with nucleotides and genomic DNA. The binding of DNA and various nucleotide species (polyAU, UMP Na2, ADP Na3, dATP Na, AMP, uridine, ATP Mg) by halloysite nanotubes was tested using UV-spectroscopy. The study revealed that binding of different nucleotides to the nanoclay varied but was low both in the presence and absence of MgCl2, while MgCl2 facilitated significantly the binding of longer molecules such as DNA and polyAU. Modification of the nanotubes with DNA and nucleotide species was further confirmed by measurements of ζ-potentials. DNA-Mg-modified nanotubes were characterized using transmission electron (TEM), atomic force (AFM) and hyperspectral microscopies. Thermogravimetric analysis corroborated the sorption of DNA by the nanotubes, and the presence of DNA on the nanotube surface was indicated by changes in the surface adhesion force measured by AFM. DNA bound by halloysite in the presence of MgCl2 could be partially released after addition of phosphate buffered saline. DNA binding and release from halloysite nanotubes was tested in the range of MgCl2 concentrations (10–100 mM). Even low MgCl2 concentrations significantly increased DNA sorption to halloysite, and the binding was leveled off at about 60 mM. DNA-Mg-modified halloysite nanotubes were used for obtaining a regular pattern on a glass surface by evaporation induced self-assembly process. The obtained spiral-like pattern was highly stable and resisted dissolution after water addition. Our results encompassing modification of non-toxic clay nanotubes with a natural polyanion DNA will find applications for construction of gene delivery vehicles and for halloysite self-assembly on various surfaces (such as skin or hair).

Bindel-PCR: a novel and convenient method for identifying CRISPR/Cas9-induced biallelic mutants through modified PCR using Thermus aquaticus DNA polymerase

We developed a novel and convenient method for rapidly identifying CRISPR/Cas9-based genome-edited biallelic knockout (KO) cells/individuals carrying insertions or deletions of a few nucleotides (indels) by performing PCR on genomic DNA samples under stringent conditions and low MgCl2 concentrations. The biallelic KO samples can be judged as ‘negative’ under these conditions. The sense primer corresponds to the sequence recognised by guide RNA and subsequently cleaved by Cas9 immediately upstream of a target gene’s proto-spacer adjacent motif (PAM), and the reverse primer corresponds to the sequence ~200 bp downstream from the PAM. PCR performed using this primer set under standard MgCl2 concentrations (1.5–2.5 mM) should generate PCR products derived from both mutated and unedited alleles, whereas PCR performed using lower MgCl2 concentrations (0.8–2 mM) should yield products derived from unedited alleles. This enables high-throughput screening of biallelic mutants among cells/embryos having ≥1 indels at a region within 5 bp upstream of the PAM (where more than 94% of indels are known to appear). We performed proof-of-principle analyses of this novel approach using genome-edited Et1, Tyr, Ramp1, Ramp3, and Rosa26 mouse samples carrying various types of indels, and demonstrate that this new technique allows rapid identification of biallelic KO mutants among samples carrying various types of indels and mosaic mutations with 100% accuracy. We name this system detection of biallelic KO mutants harbouring indels using PCR (Bindel-PCR).

Excimer based fluorescent pyrene-ferritin conjugate for protein oligomerization studies and imaging in living cells.

Ferritin self-assembly has been widely exploited for the synthesis of a variety of nanoparticles for drug-delivery and diagnostic applications. However, despite the crucial role of ferritin self-assembly mechanism for probes encapsulation, little is known about the principles behind the oligomerization mechanism. In the present work, the novel “humanized” chimeric Archaeal ferritin HumAfFt, displaying the transferrin receptor-1 (TfR1) recognition motif typical of human H homopolymer and the unique salt-triggered oligomerization properties of Archaeoglobus fulgidus ferritin (AfFt), was site-selectively labeled with N-(1-pyrenyl)maleimide on a topologically selected cysteine residue inside the protein cavity, next to the dimer interface. Pyrene characteristic fluorescence features were exploited to investigate the transition from a dimeric to a cage-like 24-meric state and to visualize the protein in vitro by two photon fluorescence microscopy. Indeed, pyrene fluorescence changes upon ferritin self-assembly allowed to establish, for the first time, the kinetic and thermodynamic details of the archaeal ferritins oligomerization mechanism. In particular, the magnesium induced oligomerization proved to be faster than the monovalent cation-triggered process, highly cooperative, complete at low MgCl2 concentrations, and reversed by treatment with EDTA. Moreover, pyrene intense excimer fluorescence was successfully visualized in vitro by two photon fluorescence microscopy as pyrene-labeled HumAfFt was actively uptaken into HeLa cells by human transferrin receptor TfR1 recognition, thus representing a unique nano-device building block for two photon fluorescence cell imaging.

Reversible changes of canavalin solubility controlled by divalent cation concentration in crude sword bean extract

Canavalin is a vicilin-class (7S) storage protein found in sword bean (Canavalia gladiata). Our previous report indicated that canavalin is precipitated by the addition of 20mMMgCl2 to crude sword bean extract. Here, we examined the solubility changes induced by the addition of Mg2+ and Ca2+ at various concentrations. Canavalin tended to be insolubilized at relatively low concentrations of MgCl2 (<20mM) and solubilized at relatively high concentrations (>20mM). In addition, canavalin was slightly insolubilized in the presence of NaCl. Overall, the results revealed that solubility changes are reversible and depend on the concentration of divalent cations. Therefore, we suggested a reaction scheme that describes the effects of divalent cations on the solubility of canavalin, which would facilitate the study of its physiological function and the application of canavalin in the food processing industry.

Alignment of Gold Nanoparticle-Decorated DNA Origami Nanotubes: Substrate Prepatterning versus Molecular Combing

DNA origami has become an established technique for designing well-defined nanostructures with any desired shape and for the controlled arrangement of functional nanostructures with few nanometer resolution. These unique features make DNA origami nanostructures promising candidates for use as scaffolds in nanoelectronics and nanophotonics device fabrication. Consequently, a number of studies have shown the precise organization of metallic nanoparticles on various DNA origami shapes. In this work, we fabricated large arrays of aligned DNA origami decorated with a high density of gold nanoparticles (AuNPs). To this end, we first demonstrate the high-yield assembly of high-density AuNP arrangements on DNA origami adsorbed to Si surfaces with few unbound background nanoparticles by carefully controlling the concentrations of MgCl2 and AuNPs in the hybridization buffer and the hybridization time. Then, we evaluate two methods, i.e., hybridization to prealigned DNA origami and molecular combing in a receding meniscus, with respect to their potential to yield large arrays of aligned AuNP-decorated DNA origami nanotubes. Because of the comparatively low MgCl2 concentration required for the efficient immobilization of the AuNPs, the prealigned DNA origami become mobile and displaced from their original positions, thereby decreasing the alignment yield. This increased mobility, on the other hand, makes the adsorbed origami susceptible to molecular combing, and a total alignment yield of 86% is obtained in this way.

Synergy effects of ions, resin, and asphaltene on interfacial tension of acidic crude oil and low–high salinity brines

It is well established that the heavy oil components including asphaltenes and resins play vital roles on the interfacial tension (IFT) of acidic crude oil (ACO) and aqueous solutions. Therefore, this experimental work is designed to investigate the possible synergism between salt ions, resin, and asphaltene on the IFT of ACO/low and high salinity brines containing MgCl2/NaCl and CaCl2. The results demonstrate that a complex ion of MgCl2 – resin component created in the solution could occupy the sites at the interface at high MgCl2 concentration. However, the results show that on the contrary, the molecular arrangement of MgCl2 and asphaltene at low and high MgCl2 concentration could be broken as the CaCl2 is able to hackle the molecular management due to the high affinity of Ca2+ to asphaltene molecules compared to that of Mg2+.

Effects of Cosolvents on the Folding and Catalytic Activities of the Hammerhead Ribozyme.

The RNA cleavage activity of the hammerhead ribozyme has been compared in various mixed aqueous solutions containing cosolvents. Kinetic analysis revealed that the tested cosolvents enhanced the ribozyme activity, particularly at low MgCl2 concentrations. These enhancements, in some cases of more than tenfold, resulted from a reduction in the Mg(2+) concentration required for substrate cleavage. An inverse correlation was found between the MgCl2 concentration essential for efficient catalysis and the dielectric constant values. In contrast, FRET measurements showed no substantial influence of cosolvents on the Mg(2+) -induced structural transitions. The results suggest that the solution environment has various effects on the Mg(2+) interactions involved in the catalysis and global folding of the ribozyme.

Enhanced cellulose fermentation and end-product synthesis by Clostridium thermocellum with varied nutrient compositions under carbon-excess conditions

Sixteen combinations of seven growth nutrients, namely α-cellulose, yeast extract (YE), urea, CaCl2·2H2O, MgCl2·6H2O, FeSO4·6H2O and vitamins, were studied to improve direct cellulose fermentation by Clostridium thermocellum DSM 1237 under carbon-excess conditions. Varied nutrient compositions improved cellulose fermentation conditions for C. thermocellum and displayed two major types of effects: a general growth enhancement effect and a carbon-flux shifting effect. Different concentrations of the four most influential nutrients (YE, α-cellulose, CaCl2 and MgCl2) resulted in enhanced yields of ethanol or H2. High ethanol yields, high ethanol to acetate (E/A) ratios, and low acetate and H2 yields were obtained when YE, α-cellulose and CaCl2 were present at high concentrations, in combination with low concentrations of magnesium. Vitamins were identified as a relatively less influential nutrient, but high concentrations of vitamins supported enhanced yields of acetate and H2. High urea and YE in combination with low MgCl2 concentrations enhanced cellulose utilization per unit mass of cells and cell-specific yields of both ethanol and H2. Volumetric yields for ethanol and H2 were improved by 2.3-fold (76.5 mM) and 2.04-fold (71.22 mmol per liter), respectively, compared with the basic combination. The highest hydrogen yield (1.64 mol/mol glucose) was obtained in the combination with the lowest ethanol yields while the lowest hydrogen yielding combination had the highest ethanol yield of 1.36 mol/mole glucose, representing 68% of the theoretical maximum for ethanol. The culture conditions determined by this study can be optimized further for enhanced production of either ethanol or H2 by direct cellulose fermentation.

Role of magnesium chloride on the purity and activity of ovomucin during the isolation process

Purification of ovomucin is still an empirical technique and sometimes insufficient quantities of ovomucin are purified to allow characterization. Here we aimed to investigate the effect of MgCl2 on the purity and bioactivity of ovomucin during isoelectric precipitation process and to develop an effective protocol to prepare pure ovomucin with high bioactivity. It was found that addition of MgCl2 is an alternative approach to remove lysozyme from ovomucin, and that the hemagglutination inhibition (HI) activity of ovomucin with MgCl2 against New Disease Virus (NDV) was about two times higher than the protein without salts. Thus, an improved procedure comprises a precipitation with 0.05mol/L CaCl2 followed by precipitation with 0.05mol/L MgCl2 was developed for the isolation of ovomucin. Better adhesion property of ovomucin was observed when low concentration of MgCl2 was added in the designed ELASA test, whereas the adhesion property of the pure ovomucin without salts to NDV was lower. Thus, magnesium (II) plays an important role in the activity of ovomucin, and the alternative method developed in this study may significantly facilitate the further research on the mechanism of ovomucin activity.

Growth characteristics, phytate contents, and coagulation properties of soymilk from a low-phytate Japanese soybean (Glycine max (L.) Merr.) line

The phytic acid (myo-inositol hexakisphosphate or InsP6) content of seed crops is important to their nutritional quality. Since it represents 75 ± 10% of the total seed phosphorus (P), phytic acid is also important regarding the management of P in agricultural production. A low-phytate F5 line, No. T-2-250-4-20, was selected from the progeny of a cross between the low-phytate soybean line CX1834 and the Japanese commercial cultivar Tanbakuro. This line and its parents were grown in a field nursery, and the growth characteristics, phytate accumulation, and processing suitability for tofu were evaluated. At full maturity, the weight of seeds per plant of line T-2-250-4-20 was 5.2- and 1.3-fold higher than that of CX1834 and Tanbakuro, respectively. The amount of phytate-phosphorus as a percentage of the total P content in seeds was 23% in line T-2-250-4-20-34, 30% in CX1834, and 69% in Tanbakuro. No significant difference was observed among the three cultivars/lines in their seed magnesium (Mg), potassium (K), crude protein, and sugar. However, the calcium (Ca), crude fat and ash contents in seeds of line T-2-250-4-20-34 and Tanbakuro was lowered compared to that of CX1834. The breaking stress of tofu was estimated employing a rheometer with a decreasing concentration of the coagulant magnesium chloride (MgCl2), starting at 15.7 mmol L−1. In tofu made from Tanbakuro, the concentration of MgCl2 required to achieve the maximum breaking stress was 12.6 mmol L−1; however, it was 9.5 mmol L−1 for tofu made from T-2-250-4-20-34 and CX1834. The tofu made from Tanbakuro was soft and broke at 6.3 mmol L−1 MgCl2, but, in line T-2-250-4-20-34, harder tofu was made with lower MgCl2 concentrations. No difference was observed among the cultivars/lines in the SDS-PAGE patterns of protein in soymilk. These results indicate that we have developed a low-phytate soybean with adequate productivity, and confirmed that tofu made from the low-phytate T-2-250-4-20-34 soybean becomes coagulated and harder at a lower MgCl2 concentration than that from high-phytate soybean cultivars.

Effect of MgCl&lt;sub&gt;2&lt;/sub&gt; on Germination, Growth and Biomass Allocation of the Radish CV. "Cherry Belle"

Problem statement: To determine the effect of MgCl2 on radish (Raphanus sativus), plants were grown in controlled environmental growth chambers and given one of three watering treatments; 0.01, 0.1 and 0 M (control) MgCl2. Approach: At high concentrations (0.1 M), MgCl2 was toxic resulting in very low (44%) germination rates and the subsequent death of all but three undersized plants over the 28 day growth period. Results: At lower concentrations (0.01 M), MgCl2 resulted in enhanced germination rates (5.55%) and a mass increase (39.54%) over the control. Conclusion/Recommendations: While low concentrations of MgCl2 resulted in an increase in total biomass, biomass allocation was shifted largely from roots to shoots (55.88% higher root/shoot ratio in the control).

Structural Diversity of Triplet Repeat RNAs

Tandem repeats of various trinucleotide motifs are present in the human transcriptome, but the functions of these regular sequences, which likely depend on the structures they form, are still poorly understood. To gain new insight into the structural and functional properties of triplet repeats in RNA, we have performed a biochemical structural analysis of the complete set of triplet repeat transcripts, each composed of a single sequence repeated 17 times. We show that these transcripts fall into four structural classes. The repeated CAA, UUG, AAG, CUU, CCU, CCA, and UAA motifs did not form any higher order structure under any analyzed conditions. The CAU, CUA, UUA, AUG, and UAG repeats are ordered according to their increasing tendency to form semistable hairpins. The repeated CGA, CGU, and all CNG motifs form fairly stable hairpins, whereas AGG and UGG repeats fold into stable G-quadruplexes. The triplet repeats that formed the most stable structures were characterized further by biophysical methods. UV-monitored structure melting revealed that CGG and CCG repeats form, respectively, the most and least stable hairpins of all CNG repeats. Circular dichroism spectra showed that the AGG and UGG repeat quadruplexes are formed by parallel RNA strands. Furthermore, we demonstrated that the different susceptibility of various triplet repeat transcripts to serum nucleases can be explained by the sequence and structural features of the tested RNAs. The results of this study provide a comprehensive structural foundation for the functional analysis of triplet repeats in transcripts.

Folding of Bacterial Group I Ribozyme in Crowded Solutions

Crowder molecules in solution alter the equilibrium between folded and unfolded states of biological macromolecules. It is therefore critical to account for the influence of these other molecules when describing the folding of RNA inside the cell, which contains up to 30% by volume of macromolecules.In this contribution we report the results of small angle x-ray scattering experiments on a catalytic RNA, or ribozyme, from the bacterium Azoarcus sp BH72 in the presence of polyethylene-glycol 1000 (PEG-1000), a molecular crowder with average molecular weight 1000 Da. We observe that, in agreement with expected excluded volume effects, the presence of PEG shifts the equilibrium to favor more compact RNA structures. Two observations highlight the stabilization of the compact RNA: firstly, the transition from the unfolded to folded (more compact) state occurs at lower MgCl2 concentrations in PEG; secondly, the radius of gyration of the unfolded RNA decreases from 76 A to 64 A as the PEG concentration increases from 0 to 20 % wt./vol.We discuss several physical effects by which the crowder molecules can possibly influence the RNA folding. These include modification of ionic activity, modification of water activity, and the excluded volume effect. We present evidence that whilst all of the above effects probably change the RNA folding with respect to a dilute aqueous solution, the shift in Mg2+ dependence of folding transition cannot be explained entirely by the change in ion activity. We conclude that the dominant influence is likely to be the excluded volume effect.

Lack of nucleosomal structure in a DNase-I-solubilized transcriptionally active chromatin fraction of Physarum polycephalum.

Light treatment of nuclei of Physarum polycephalum microplasmodia with DNase I, at low MgCl2 concentration (less than or equal to 3% DNA acid solubility, 0.1 mM MgCl2) selectively solubilizes a defined fraction of chromatin, in the form of a macromolecular complex. This fraction (up to 15% of the total chromatin) contains a full complement of the core histones and a reduced amount of histone H1, and is enriched in the high-mobility-group type of proteins. It is preferentially associated with nascent RNA and RNA polymerase B actively engaged in transcription. Digestion of DNAase-I-solubilized chromatin by micrococcal nuclease releases a size-heterogeneous population of cleavage products, indicative of lack of a typical nucleosomal packaging. It is concluded that the procedure used allows the isolation of structurally and functionally distinct regions of Physarum chromatin.

Stimulation effects of salts on growth in suspension culture of a mangrove plant, Sonneratia alba, compared with another mangrove, Bruguiera sexangula and non-mangrove tobacco BY-2 cells

Effects of a wide range of concentrations of four salts (NaCl, KCl, MgCl2, CaCl2) on cell proliferation were investigated in suspension culture of a mangrove plant, Sonneratia alba. The results were compared to those obtained with suspension culture of another mangrove species, Bruguiera sexangula and non-mangrove tobacco BY-2 cells. Effects of salts on suspension cells were determined with both large scale (LS) method using a 100 ml culture flask, and/or a small scale (SS) method using a 24-well culture plate. Settled cell volume (SCV), packed cell volume (PCV), wet weight (WW) and dry weight (DW) were measured in LS method and PCV was in SS method. High concentrations of NaCl (25–100 mM) and MgCl2 (25 mM) showed strong stimulatory effects on growth in S. alba suspension culture, and low concentrations of KCl, MgCl2 and CaCl2 (10 mM) also showed similar stimulatory effects. In contrast, a remarkable stimulatory effect was not observed in B. sexangula and tobacco BY-2 cells. Cationic ions are effective for the growth of suspension cells in both mangrove species S. alba and B. sexangula. The SS method using multi-well culture plate and micro-tube measurement method developed in this report, can be applied for analysis of factors for stress tolerance.

Variation in the phytic acid content of soybeans and its effect on consistency of tofu made from soybean varieties with high protein content

AbstractThe relationships of contents of phytic acid and protein to the breaking stress of tofu were examined using soybeans of three Japanese varieties with high protein content harvested in different locations from the years 2001 to 2003. The phytic acid content was more variable than protein content within different cultivating locations and years for the same variety and showed significant negative correlation to the breaking stress of tofu made with 0.25% MgCl2 for all the varieties (P &lt; 0.001, r = −0.60). In particular, Sachiyutaka variety was characterized by the highest negative correlation between the breaking stress of tofu and the phytic acid content in soymilk (P &lt; 0.001, r = −0.73). As MgCl2 concentration increased, the correlation between the phytic acid content and the breaking stress of tofu was reduced, and finally there was no significant correlation between them when the breaking stress reached a maximum at the particular MgCl2 concentration which is probably concerned with varietial characteristics of soybeans. These results support the idea that the variation in the content of phytic acid, which can act as a buffer against coagulation of soy protein, accounts for the fluctuation in consistency of tofu made with lower MgCl2 concentrations (ca. 0.25%) using soybeans of the same variety with different cultivating conditions. Copyright © 2005 Society of Chemical Industry

Divalent cation induced changes in structural properties of the dimeric enzyme glucose oxidase: dual effect of dimer stabilization and dissociation with loss of cooperative interactions in enzyme monomer.

Glucose oxidase (GOD) from Aspergillus niger is a dimeric enzyme having high localization of negative charges on the enzyme surface and at the dimer interface. The monovalent cations induce compaction of the native conformation of GOD and enhance stability against thermal and urea denaturation [Ahmad et al. (2001) Biochemistry 40, 1947-1955]. In this paper we report the effect of the divalent cations Ca2+ and Mg2+ on the structural and stability properties of GOD. A divalent cation concentration dependent change in native conformation and subunit assembly of GOD was observed. Low concentration (up to 1 M) of CaCl2 or MgCl2 induced compaction of the native conformation of GOD, and the enzyme showed higher stability as compared to the native enzyme against urea denaturation. However, higher concentration (> or =2.0 M) of CaCl2 or MgCl2 induced dissociation of the native dimeric enzyme, resulting in stabilization of the enzyme monomer. An interesting observation was that the 3 M CaCl2-stabilized monomer of GOD retained about 70% secondary structure present in the native GOD dimer; however, there was a complete loss of cooperative interactions between these secondary structural elements present in the enzyme. Regarding the mechanism of divalent cation induced structural changes in GOD, the studies suggest that organization of water molecules by divalent cation results in stabilization of enzyme at low divalent cation concentration, whereas direct binding of these cations to the enzyme, at higher divalent cation concentration, results in dissociation and partial unfolding of the dimeric enzyme molecule.

Magnesium-dependent Association and Folding of Oligonucleosomes Reconstituted with Ubiquitinated H2A

The MgCl2-induced folding of defined 12-mer nucleosomal arrays, in which ubiquitinated histone H2A (uH2A) replaced H2A, was analyzed by quantitative agarose gel electrophoresis and analytical centrifugation. Both types of analysis showed that uH2A arrays attained a degree of compaction similar to that of control arrays in 2 mM MgCl2. These results indicate that attachment of ubiquitin to H2A has little effect on the ability of nucleosomal arrays to form higher order folded structures in the ionic conditions tested. In contrast, uH2A arrays were found to oligomerize at lower MgCl2 concentrations than control nucleosomal arrays, suggesting that histone ubiquitination may play a role in nucleosomal fiber association.

Major phosphorylation site (Ser55) of neurofilament L by cyclic AMP-dependent protein kinase in rat primary neuronal culture.

Ser55 of neurofilament L (NF-L) is reported to be partly phosphorylated in neurons and to be phosphorylated by cyclic AMP-dependent protein kinase (PKA). Bovine NF-L was phosphorylated by PKA in a low concentration of MgCl2 (0.3 mM) and digested by trypsin. Trypsin-digested fragments were assigned by MALDI/ TOF (matrix-assisted laser desorption and ionization/ time-of-flight) mass spectrometry. Phosphorylation sites were found at Ser41, Ser55, and Ser62 in the head region, with Ser55 considered the preferred site. A site-specific phosphorylation-dependent antibody against Ser55 rendered NF-L phosphorylated at Ser55 detectable in primary cultured rat neurons. One-hour treatment with 20 nM okadaic acid increased the phosphorylation level of Ser55, and co-treatment with 10 microM forskolin enhanced it. However, forskolin alone did not elevate the phosphorylation level. As a consequence, NF-L may be phosphorylated at Ser55 by PKA or by a PKA-like kinase in vivo; however, the phosphorylation level of Ser55 may be modulated by certain phosphatases sensitive to okadaic acid.

The simultaneous binding of two double-stranded DNA molecules by Escherichia coli RecA protein

We have characterized the double-stranded DNA (dsDNA) binding properties of RecA protein, using an assay based on changes in the fluorescence of 4′,6-diamidino-2-phenylindole (DAPI)-dsDNA complexes. Here we use fluorescence, nitrocellulose filter-binding, and DNase I-sensitivity assays to demonstrate the binding of two duplex DNA molecules by the RecA protein filament. We previously established that the binding stoichiometry for the RecA protein-dsDNA complex is three base-pairs per RecA protein monomer, in the presence of ATP. In the presence of ATPγS, however, the binding stoichiometry depends on the MgCl2 concentration. The stoichiometry is 3 bp per monomer at low MgCl2 concentrations, but changes to 6 bp per monomer at higher MgCl2 concentrations, with the transition occurring at approximately 5 mM MgCl2. Above this MgCl2 concentration, the dsDNA within the RecA nucleoprotein complex becomes uncharacteristically sensitive to DNase I digestion. For these reasons we suggest that, at the elevated MgCl2 conditions, the RecA-dsDNA nucleoprotein filament can bind a second equivalent of dsDNA. These results demonstrate that RecA protein has the capacity to bind two dsDNA molecules, and they suggest that RecA or RecA-like proteins may effect homologous recognition between intact DNA duplexes.