Direct transfer of proteins between DNA helices is a recognized important feature of the recognition site search process. Direct transfer is characterized by a dissociation rate that depends on total ...DNA concentration. This is taken as evidence for the formation of an intermediate DNA-protein-DNA ternary complex. We find that the dissociation rate of EcoRI-DNA-specific complexes at 80 mM NaCl depends on the concentration of competitor oligonucleotide suggesting that direct transfer contributes to EcoRI dissociation. This dependence on competitor DNA concentration is not seen at 180 mM salt. A careful examination of the salt concentration dependence of the dissociation rate, however, shows that the predictions for the formation of a ternary complex are not observed experimentally. The findings can be rationalized by considering that just after dissociating from a DNA fragment the protein remains in close proximity to that fragment, can reassociate with it, and diffuse back to the recognition site rather than bind to an oligonucleotide in solution, a hopping excursion. The probability that a protein will bind to an oligonucleotide during a hop can be approximately calculated and shown to explain the data. A dependence of the dissociation rate of a DNA-protein complex on competitor DNA concentration does not necessarily mean direct transfer.
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•The early Paleozoic Julia deposit comprises oxidized-type, garnet-dominant skarn with bornite and chalcopyrite followed by quartz-carbonate-sulfide veins with Cu-Au-Bi-Te ...mineralization.•The deposit is related to a post-collisional high-K calc-alkaline to shoshonitic igneous suite.•U-Pb zircon data indicate the Late Cambrian age of causative igneous rocks.•Long-lasting (20 m.y) magmatic evolution culminated in rapid emplacement (5 m.y.) of mineralizing intrusions.
The small but high-grade (average 1.29% to 2.04% Cu and 0.09–0.12% Mo for different orebodies, with also significant Au contents) Julia skarn Cu-Au-Mo deposit is situated some 30 km northeast from the Sorskoe porphyry Mo-Cu deposit in the Kuznetsk Alatau, a Paleozoic terrane in the northernmost part of the Altai-Sayan orogenic system, part of the Central Asian Orogenic Belt. The deposit comprises prograde calcic skarns and later retrograde skarn characterized by abundant andradite-rich garnet defining their oxidized type. These alteration assemblages bear abundant Cu sulfides (chalcopyrite and bornite), which are associated with magnetite and are partially overprinted by weak propylitic and stronger phyllic (carbonate-phyllic) alteration assemblages. Molybdenite is present in these assemblages, and various sulfides and sulfosalts, tellurides, and Bi minerals, as well as native Au are associated with phyllic alteration.
The deposit is related to a magnetite-series, I-type, multiphase monzonite-syenite(-quartz syenite)-quartz monzonite-monzogranite igneous complex. Isotopic U-Pb zircon data indicate a Late Cambrian age (∼505 to 485 Ma) of the igneous rocks, whereas their geochemical signatures correspond to high-K calc-alkaline to shoshonitic intrusions emplaced in a post-collisional environment, with possible relationships to a subducted slab break-off. Magmatic evolution included a generation of shoshonitic magma by a low-degree partial melting of the metasomatically-enriched upper mantle, followed by amphibole fractionation in a deep (lower crustal?) magma chamber and then by magma fractionation and emplacement at shallower crustal levels. Although the overall magmatic evolution of the parental intrusions was long-lasting (ca. 20 m.y.), the mineralization is associated with a relatively rapid (ca. 5 m.y.) emplacement of more differentiated granitoid intrusions as well as late mafic dikes.
The deposit represents an early Paleozoic (Late Cambrian) Cu-Au(-Mo) mineralization in the Altai-Sayan orogenic system and the broader Central Asian Orogenic Belt, thus confirming the Late Cambrian (to Early Ordovician) Cu-Au-Mo metallogenic event in the region. This, together with the broad regional occurrence of the Cambrian-Early Ordovician igneous complexes and related porphyry/skarn Cu-Au(-Mo) mineralization, emphasizes the early Paleozoic porphyries/skarns as a major regional event prior to much better known late Paleozoic porphyries/skarns in the Central Asian Orogenic Belt. There is a potential of the early Paleozoic terranes to host much more significant Cu-Au(-Mo) mineralization including that of skarn, porphyry and associated types.
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•The Glafirinskoe and related deposits belong to a regional Early Paleozoic belt of Cu-Au-Mo skarn and porphyry deposits.•The deposits are related to post-collisional high-K ...calc-alkaline to shoshonitic intrusions.•Isotopic U-Pb zircon dating reveals Early Ordovician ages of the parental intrusions.•The deposits comprise W and/or Mo mineralization defining their Cu-Au(-Mo-W) metallogenic type.•Mineralization was formed by high- to low-salinity aqueous-chloride and aqueous-carbonic fluids.
The Glafirinskoe and related skarn Cu-Au (+Mo, W) deposits represent an Early Paleozoic (Late Cambrian to very early Ordovician) mineralization in the Altaid Belt, also known as the Central Asian Orogenic Belt. They are associated with multiphase plutons of high-K calc-alkaline to shoshonitic rocks that are related to a low-degree partial melting of metasomatically-enriched upper mantle, followed by amphibole fractionation in a deep (lower crustal ?) magma chamber and then by magma fractionation and emplacement at shallower crustal levels. Isotopic U-Pb zircon dating of the igneous rocks yields early Ordovician (ca. 480–478 Ma and 483 Ma) age. These rocks were emplaced in a continental magmatic arc after the Late Cambrian collision, in a post-collisional setting involving the stall and break-off of the subducting slab during the Late Cambrian to Early Ordovician.
The deposits comprise prograde and retrograde skarns that are characterized by abundant andradite-rich garnet, which defines their oxidized type. Wider zones of potassic (quartz-K-feldspar) and calc-potassic (quartz-K-feldspar-calcite-garnet) alteration occur in the igneous rocks, with their further overprint by propylitic (with abundant scapolite) and phyllic (carbonate-phyllic) alteration. All these alteration assemblages bear abundant chalcopyrite and minor bornite. Phyllic alteration assemblages also contain various As, Sb, Pb, Zn, Co, Ni sulfides and sulfosalts, tellurides, Bi minerals, and native Au. Scheelite and/or molybdenite are locally present in some mineralized zones. The Cu-Au-Mo(-W) mineralization occurs as skarn orebodies that are spatially associated with porphyry-style alteration assemblages reflecting the high exploration potential for additional, but concealed, skarn-porphyry Cu-Au-Mo deposits in the vicinity.
The retrograde skarn and potassic (calc-potassic) alteration assemblages were formed from high temperature (570–580 °C), high salinity, aqueous chloride magmatic-hydrothermal fluid, with its boiling and phase separation indicating hydrostatic conditions at a relatively deep (~6 km) skarn-porphyry environment. Propylitic alteration assemblages were formed at lower temperatures (~380–420 °C) from a Ca-Na-chloride, low salinity (~8 wt% NaCl-eq.) magmatic-hydrothermal fluid. Fluid inclusions in minerals from phyllic alteration assemblages indicate the influx of a CO2-rich fluid at about 270–290 °C and 1400 ± 50 bars and subsequent mixing with more saline (20 wt% NaCl-eq.) and cooler (~240 °C) hydrothermal fluids.
Hydrothermal synthesis experiments were performed in order to quantify the states of Au and As in pyrite and marcasite. The experiments were performed at 350 °C/500 bar and 490 °C/1000 bar ...(pyrite–pyrrhotite buffer, C(NaCl) = 15 and 35 wt.%). The synthesis products were studied by EPMA, LA-ICP-MS, and EBSD. The EPMA was applied for simultaneous determinations of Au, As, Fe, and S, with a Au detection limit of 45–48 ppm (3σ). The analyses were performed along profiles across zonal grains. The concentrations of As and Au up to 5 wt.% and 8000 ppm, respectively, were determined in pyrite and up to 6 wt.% and 1300 ppm in marcasite. In pyrite, the Au concentration decreases with fluid salinity and temperature increases. Strong positive Au–As correlation and strong negative Au–Fe and As–S correlation were identified in pyrite. Comparison of the correlations with theoretical lines implies Au–As clustering. The cluster stoichiometry is inferred to be AuAs10. Most probably, As in pyrite presents in the form of clusters and in the As→S solid solution. Incorporation of Au in As-rich pyrite can be controlled by the reductive deposition mechanism. In marcasite, the concentrations of Au are not correlated with the As content. The AuAs10 clusters enrich the {210}, {113}, and {111} pyrite faces, where the former exhibits the highest affinity to Au and As. The affinity of {110} and {100} forms to Au and As is lower. Implication of the experimental results to data for natural auriferous pyrite shows that the increase of Au content at C(As) > 0.5–1 wt.% is caused by the incorporation of the Au-As clusters, but not because of the formation of Au→Fe solid solution. Therefore, the concentration of “invisible” gold in pyrite is dictated solely by the hydrothermal fluid chemistry and subsequent ore transformations.
Many specific sequence DNA binding proteins locate their target sequence by first binding to DNA nonspecifically, then by linearly diffusing or hopping along DNA until either the protein dissociates ...from the DNA or it finds the recognition sequence. We have devised a method for measuring one-dimensional diffusion along DNA based on the ratio of the dissociation rate of protein from DNA fragments containing one specific binding site to the dissociation rate from DNA fragments containing two specific binding sites. Our extensive measurements of dissociation rates and specific–nonspecific relative binding constants of the restriction nuclease EcoRI enable us to determine the diffusion rate of nonspecifically bound protein along the DNA. By varying the distance between the two binding sites, we confirm a linear diffusion mechanism. The sliding rate is relatively insensitive to salt concentration and osmotic pressure, indicating that the protein moves smoothly along the DNA probably following the helical phosphate-sugar backbone of DNA. We calculate a diffusion coefficient for EcoRI of 3
×
10
4 bp
2 s
−
1 EcoRI is able to diffuse ∼
150 bp, on average, along the DNA in 1 s. This diffusion rate is about 2000-fold slower than the diffusion of free protein in solution. A factor of 40–50 can be accounted for by rotational friction resulting from following the helical path of the DNA backbone. Two possibilities could account for the remaining activation energy: salt bridges between the DNA and the protein are transiently broken, or the water structure at the protein–DNA interface is disrupted as the two surfaces move past each other.
The electrophoretic mobility-shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA-protein interactions. EMSA, as standardly practiced today, works well for ...complexes with association binding constants Ka>10⁹ M⁻¹ under normal conditions of salt and pH. Many DNA-protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using EMSA. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein-DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and CE is straightforward.
The DNA binding stringency of restriction endonucleases is crucial for their proper function. The X-ray structures of the specific and non-cognate complexes of the restriction nuclease EcoRV are ...considerably different suggesting significant differences in the hydration and binding free energies. Nonetheless, the majority of studies performed at pH 7.5, optimal for enzymatic activity, have found a < 10-fold difference between EcoRV binding constants to the specific and nonspecific sequences in the absence of divalent ions. We used a recently developed self-cleavage assay to measure EcoRV-DNA competitive binding and to evaluate the influence of water activity, pH and salt concentration on the binding stringency of the enzyme in the absence of divalent ions. We find the enzyme can readily distinguish specific and nonspecific sequences. The relative specific-nonspecific binding constant increases strongly with increasing neutral solute concentration and with decreasing pH. The difference in number of associated waters between specific and nonspecific DNA-EcoRV complexes is consistent with the differences in the crystal structures. Despite the large pH dependence of the sequence specificity, the osmotic pressure dependence indicates little change in structure with pH. The large osmotic pressure dependence means that measurement of protein-DNA specificity in dilute solution cannot be directly applied to binding in the crowded environment of the cell. In addition to divalent ions, water activity and pH are key parameters that strongly modulate binding specificity of EcoRV.
A significant part of the primary gold reserves in the world is contained in sulphide ores, many types of which are refractory in gold processing. The deposits of refractory sulphide ores will be the ...main potential source of gold production in the future. The refractory gold and silver in sulphide ores can be associated with micro- and nano-sized inclusions of Au and Ag minerals as well as isomorphous, adsorbed and other species of noble metals (NM) not thoroughly investigated. For gold and gold-bearing deposits of the Urals, distribution and forms of NM were studied in base metal sulphides by laser ablation-inductively coupled plasma mass spectrometry and by neutron activation analysis. Composition of arsenopyrite and As-pyrite, proper Au and Ag minerals were identified using electron probe microanalysis. The ratio of various forms of invisible gold—which includes nanoparticles and chemically bound gold—in sulphides is discussed. Observations were also performed on about 120 synthetic crystals of NM-doped sphalerite and greenockite. In VMS ores with increasing metamorphism, CAu and CAg in the major sulphides (sphalerite, chalcopyrite, pyrite) generally decrease. A portion of invisible gold also decreases —from ~65–85% to ~35–60% of the total Au. As a result of recrystallisation of ores, the invisible gold is enlarged and passes into the visible state as native gold, Au-Ag tellurides and sulphides. In the gold deposits of the Urals, the portion of invisible gold is usually <30% of the bulk Au.
Using the osmotic stress technique together with a self-cleavage assay we measure directly differences in sequestered water between specific and nonspecific DNA-BamHI complexes as well as the numbers ...of water molecules released coupled to specific complex formation. The difference between specific and nonspecific binding free energy of the BamHI scales linearly with solute osmolal concentration for seven neutral solutes used to set water activity. The observed osmotic dependence indicates that the nonspecific DNA-BamHI complex sequesters some 120–150 more water molecules than the specific complex. The weak sensitivity of the difference in number of waters to the solute identity suggests that these waters are sterically inaccessible to solutes. This result is in close agreement with differences in the structures determined by x-ray crystallography. We demonstrate additionally that when the same solutes that were used in competition experiments are used to probe changes accompanying the binding of free BamHI to its specific DNA sequence, the measured number of water molecules released in the binding process is strikingly solute-dependent (with up to 10-fold difference between solutes). This result is expected for reactions resulting in a large change in a surface exposed area.
The binding of the restriction endonuclease
EcoRI to DNA is exceptionally specific. Even a single basepair change (“star” sequence) from the recognition sequence, GAATTC, decreases the binding free ...energy of
EcoRI to values nearly indistinguishable from nonspecific binding. The difference in the number of waters sequestered by the protein-DNA complexes of the “star” sequences TAATTC and CAATTC and by the specific sequence complex determined from the dependence of binding free energy on water activity is also practically indistinguishable at low osmotic pressures from the 110 water molecules sequestered by nonspecific sequence complexes. Novel measurements of the dissociation rates of noncognate sequence complexes and competition equilibrium show that sequestered water can be removed from “star” sequence complexes by high osmotic pressure, but not from a nonspecific complex. By 5 Osm, the TAATTC “star” sequence complex has lost almost 90 of the ∼110 waters initially present. It is more difficult to remove water from the CAATTC “star” sequence complex. The sequence dependence of water loss correlates with the known sequence dependence of “star” cleavage activity.
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