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Full chemical equilibrium calculations of the sequence of condensation of the elements from cosmic gases made by total vaporization of dust-enriched systems were performed to investigate the oxidation state of the resulting condensates. Computations included 23 elements and 374 gas species over a range of -3=log10(total P) to -6 bar and for enrichments to 1000x in dust of C1 chondritic composition relative to a system of solar composition. Because liquids are stable condensates in these systems, the MELTS non-ideal solution model for silicate liquids was used. Condensation at logP=-3 bar and dust enrichments of 100x, 500x and 1000x occurs at oxygen fugacities of IW-3.1, IW-1.7 and IW-1.2, respectively, and, at the temperature of cessation of direct condensation of olivine from the vapor, yields X(fayalite) of 0.019, 0.088 and 0.164, respectively. Silicate liquid is a stable condensate at dust enrichments >~12.5x at logP=-3. At 1000x, the Na and K oxide contents of the last liquid reach 10.1 and 1.3 wt%, respectively, at logP=-3 bar. At logP=-3 bar, iron sulfide liquids are stable condensates at dust enrichments at least as low as 500x, and the predicted distribution of Fe between metal, silicate and sulfide at 1310K and a dust enrichment of 560x matches that found in H chondrites, and at 1330K and 675x matches that of L chondrites prior to metal loss. With some exceptions, many chondrule glass compositions fall along bulk composition trajectories for liquids in equilibrium with cosmic gases at logP=-3 bar and dust enrichments between 600x and 1000x. If these chondrules formed by secondary melting of mixtures of condensates that formed at different T, nebular regions with characteristics such as these would have been necessary to prevent loss of Na by evaporation and FeO by reduction from the liquid precursors, assuming that liquids and gas were hot for enough time to have equilibrated., 58 pages, 19 figures, 8 tables

The degree to which dust enrichment enhances the oxygen fugacity (fO2) of a system otherwise solar in composition depends on the dust composition. Equilibrium calculations were performed at 10−3 bar in systems enriched by a factor of 104 in two fundamentally different types of dust to investigate the iron oxidation state in both cases. One type of dust, called SC for solar condensate, stopped equilibrating with solar gas at too high a temperature for FeO or condensed water to be stabilized in any form, and thus has the composition expected of a nebular condensate. The other has CI chondrite composition, appropriate for a parent body that accreted from SC dust and low-temperature ice. Upon total vaporization at 2300 K, both systems have high fO2, >IW. In the SC dust-enriched system, FeO of the bulk silicate reaches ~10 wt% at 1970 K but decreases to

The silicate and metal clasts in CB chondrites have been inferred to form as condensates from an impact-generated vapor plume between a metal-rich body and a silicate body. A detailed study of the condensation of impact-generated vapor plumes showed that the range of CB silicate clast compositions could not be successfully explained without invoking a chemically differentiated target. Here, we report the most comprehensive elemental study yet performed on CB silicates with 32 silicate clasts from nine slices of Gujba analyzed by laser ablation inductively coupled plasma mass spectrometry for 53 elements. Like in other studies of CBs, the silicate clasts are either barred olivine (BO) or cryptocrystalline (CC) in texture. In major elements, the Gujba silicate clasts ranged from chondritic to refractory enriched. Refractory element abundances ranged from 2t o 10� CI, with notable anomalies in Ba, Ce, Eu, and U abundances. The two most refractory-enriched BO clasts exhibited negative Ce anomalies and were depleted in U relative to Th, characteristic of volatilization residues, while other BO clasts and the CC clasts exhibited positive Ce anomalies with excess U (1–3 � CI), and Ba (1–6 � CI) anomalies indicating recondensation of ultra-refractory element depleted vapor. The Rare Earth Elements (REE) also exhibit light REE (LREE) enrichment or depletion in several clasts with a range of (La/Sm)CI of 0.9–1.8. This variation in the LREE is essentially impossible to accomplish by processes involving vapor–liquid or vapor–solid exchange of REE, and appears to have been inherited from a differentiated target. The most distinctive evidence for inherited chemical differentiation is observed in highly refractory element (Sc, Zr, Nb, Hf, Ta, Th) systematics. The Gujba clasts exhibit fractionations in Nb/Ta that correlate positively with Zr/Hf and span the range known from lunar and Martian basalts, and exceed the range in Zr/Hf variation known from eucrites. Variations of highly incompatible refractory elements (e.g., Th) against less incompatible elements (e.g., Zr, Sr, Sc) are not chondritic, but exhibit distinctly higher Th abundances requiring a differentiated crust to be admixed with depleted mantle in ratios that are biased to higher crust/mantle ratios than in a chondritic body. The possibility that these variations are due to admixture of refractory inclusion-debris into normal chondritic matter is raised but cannot be definitively tested because existing ‘‘bulk” analyses of CAIs carry artifacts of unrepresentative sampling. The inferences drawn from the compositions of Gujba silicate clasts, here, complement what has been inferred from the compositions of metallic clasts, but provide surprisingly detailed insight into the structure of the target. Evidence that metal and silicate in CB chondrites both formed from impact-generated vapor plumes, taken together with recent work on metallic nodules in E chondrites, and on ordinary chondrites, indicates that chondrule formation occurs by this mechanism quite widely. However, the nature of

The impact hypothesis for the origin of CB chondrites was tested by performing equilibrium condensation calculations in systems composed of vaporized mixtures of projectile and target materials. When one of the impacting bodies is composed of the metal from CR chondrites and the other is an H chondrite, good agreement can be found between calculated and observed compositions of unzoned metal grains in CB chondrites but the path of composition variation of the silicate condensate computed for the same conditions that reproduce the metal grain compositions does not pass through the measured compositions of barred olivine (BO) or cryptocrystalline (CC) chondrules in the CBs. The discrepancy between measured chondrule compositions and those of calculated silicates is not reduced when diogenite, eucrite or howardite compositions are substituted for H chondrite as the silicate-rich impacting body. If, however, a CR chondrite body is differentiated into core, a relatively CaO-, Al 2 O 3 -poor mantle and a CaO-, Al 2 O 3 -rich crust, and later accretes significant amounts of water, a collision between it and an identical body can produce the necessary chemical conditions for condensation of CB chondrules. If the resulting impact plume is spatially heterogeneous in its proportions of crust and mantle components, the composition paths calculated for silicate condensates at the same P tot , Ni/H and Si/H ratios and water abundance that produce good matches to the unzoned metal grain compositions pass through the fields of BO and CC chondrules, especially if high-temperature condensates are fractionated in the case of the CCs. While equilibrium evaporation of an alloy containing solar proportions of siderophiles into a dense impact plume is an equally plausible hypothesis for explaining the compositions of the unzoned metal grains, equilibrium evaporation can explain CB chondrule compositions only if an implausibly large number of starting compositions is postulated. Kinetic models applied to co-condensing metal grains and silicate droplets in a region of the plume with very similar composition, but with high cooling rate and sharply declining P tot during condensation, produce very good matches to the zoning profiles of Ir, Ni, Co and Cr concentrations and Fe and Ni isotopic compositions observed in the zoned metal grains in CB chondrites but produce very large positive δ 56 Fe in the cogenetic silicate, which are not found in the chondrules.

We present high-precision measurements of the Mg isotopic compositions of a suite of types I and II chondrules separated from the Murchison and Murray CM2 carbonaceous chondrites. These chondrules are olivine- and pyroxene-rich and have low 27 Al/ 24 Mg ratios (0.012-0.316). The Mg isotopic compositions of Murray chondrules are on average lighter (d 26 Mg ranging from 0.95& to 0.15& relative to the DSM-3 standard) than those of Murchison (d 26 Mg ranging from 1.27& to +0.77&). Taken together, the CM2 chondrules exhibit a narrower range of Mg isotopic compositions than those from CV and CB chondrites studied previously. The least-altered CM2 chondrules are on average lighter (average d 26 Mg = 0.39 0.30&, 2SE) than the moderately to heavily altered CM2 chondrules (average d 26 Mg = 0.11 0.21&, 2SE). The compositions of CM2 chondrules are consistent with isotopic fractionation toward heavy Mg being associated with the formation of secondary silicate phases on the CM2 parent body, but were also probably affected by volatilization and recondensation processes involved in their original formation. The low-Al CM2 chondrules analyzed here do not exhibit any mass-independent variations in 26 Mg from the decay of 26 Al, with the exception of two chondrules that show only small variations just outside of the analytical error. In the case of the chondrule with the highest Al/Mg ratio (a type IAB chondrule from Murchison), the lack of resolvable 26 Mg excess suggests that it either formed >1 Ma after calcium-aluminum-rich inclusions, or that its Al-Mg isotope systematics were reset by secondary alteration processes on the CM2 chondrite parent body after the decay of 26 Al.

To determine evaporation coefficients for the major gaseous species that evaporate from silicate melts, the Hertz–Knudsen equation was used to model the compositions of residues of chondrule analogs produced by evaporation in vacuum by Hashimoto [Hashimoto A. (1983) Evaporation metamorphism in the early solar nebula-evaporation experiments on the melt FeO–MgO–SiO2–CaO–Al2O3 and chemical fractionations of primitive materials. Geochem. J. 17, 111–145] and Wang et al. [Wang J., Davis A. M., Clayton R. N., Mayeda T. K., Hashimoto A. (2001) Chemical and isotopic fractionation during the evaporation of the FeO–MgO–SiO2–CaO–Al2O3–TiO2 rare earth element melt system. Geochim. Cosmochim. Acta 65, 479–494], in vacuum and in H2 by Yu et al. [Yu Y., Hewins R. H., Alexander C. M. O’D., Wang J. (2003) Experimental study of evaporation and isotopic mass fractionation of potassium in silicate melts. Geochim. Cosmochim. Acta 67, 773–786], and in H2 by Cohen et al. [Cohen B. A., Hewins R. H., Alexander C. M. O’D. (2004) The formation of chondrules by open-system melting of nebular condensates. Geochim. Cosmochim. Acta 68, 1661–1675]. Vapor pressures were calculated using the thermodynamic model of Ghiorso and Sack [Ghiorso M. S., Sack R. O. (1995) Chemical mass transfer in magmatic processes IV. A revised and internally consistent thermodynamic model for the interpolation and extrapolation of liquid–solid equilibria in magmatic systems at elevated temperatures and pressures. Contrib. Mineral. Petrol. 119, 197–212], except for the late, FeO-free stages of the Wang et al. (2001) and Cohen et al. (2004) experiments, where the CMAS activity model of Berman [Berman R. G. (1983) A thermodynamic model for multicomponent melts, with application to the system CaO–MgO–Al2O3–SiO2. Ph.D. thesis, University of British Columbia] was used. From these vapor pressures, evaporation coefficients (α) were obtained that give the best fits to the time variation of the residue compositions. Evaporation coefficients derived for Fe(g), Mg(g), and SiO(g) from the Hashimoto (1983) experiments are similar to those found by Alexander [Alexander C. M. O’D. (2004) Erratum. Meteoritics Planet. Sci. 39, 163] in his EQR treatment of the same data and also adequately describe the FeO-bearing stages of the Wang et al. (2001) experiments. From the Yu et al. (2003) experiments at 1723 K, αNa = 0.26 ± 0.05, and αK = 0.13 ± 0.02 in vacuum, and αNa = 0.042 ± 0.020, andαK = 0.017 ± 0.002 in 9 × 10−5 bar H2. In the FeO-free stages of the Wang et al. (2001) experiments, αMg and αSiO are significantly different from their respective values in the FeO-bearing portions of the same experiments and from the vacuum values obtained at the same temperature by Richter [Richter F. M., Davis A. M., Ebel D. S., Hashimoto A. (2002) Elemental and isotopic fractionation of Type B calcium-, aluminum-rich inclusions: experiments, theoretical considerations, and constraints on their thermal evolution. Geochim. Cosmochim. Acta 66, 521–540] for CMAS compositions much lower in MgO. When corrected for temperature, the values of αMg and αSiO that best describe the FeO-free stages of the Wang et al. (2001) experiments also adequately describe the FeO-free stage of the Cohen et al. (2004) H2 experiments, but αFe that best describes the FeO-bearing stage of the latter experiment differs significantly from the temperature-corrected value derived from the Hashimoto (1983) vacuum data.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to measure distributions of the siderophile elements V, Fe, Co, Ni, Mo, Ru, Rh, Pd, W, Re, Os, Ir, Pt, and Au in Fremdlinge with a spatial resolution of 15 to 25 m. A sulfide vein in a refractory inclusion in Allende (CV3-oxidized) is enriched in Rh, Ru, and Os with no detectable Pd, Re, Ir, or Pt, indicating that Rh, Ru, and Os were redistributed by sulfidation of the inclusion, causing fractionation of Re/Os and other siderophile element ratios in Allende CAIs. Fremdlinge in compact Type-A inclusions from Efremovka (CV3-reduced) exhibit subsolidus exsolution into kamacite and taenite and minimal secondary formation of V-magnetite and schreibersite. Siderophile element partitioning between taenite and kamacite is similar to that observed previously in iron meteorites, while preferential incorporation of the light PGEs (Ru, Rh, Pd) relative to Re, Os, Ir, and Pt by schreibersite was observed. Fremdling EM2 (CAI Ef2) has an outer rim of P-free metal that preserves the PGE signature of schreibersite, indicating that EM2 originally had a phosphide rim and lost P to the surrounding inclusion during secondary processing. Most Fremdlinge have chondrite-normalized refractory PGE patterns that are unfractionated, with PGE abundances derived from a small range of condensation temperatures, 1480 to 1468 K at Ptot 10 3 bar. Some Fremdlinge from the same CAI exhibit sloping PGE abundance patterns and Re/Os ratios up to 2 CI that likely represent mixing of grains that condensed at various temperatures. Copyright © 2003 Elsevier Ltd

The volatilization kinetics of single crystal α-SiC, polycrystalline β-SiC, and SiO_2 (cristobalite or glass) were determined in H_2-CO_2, CO-CO_2, and H_2-CO-CO_2 gas mixtures at oxygen fugacities between 1 log unit above and 10 log units below the iron-wustite (IW) buffer and temperatures in the range 1151 to 1501°C. Detailed sets of experiments on SiC were conducted at 2.8 and 6.0 log units below IW (IW-2.8 and IW-6.0) at a variety of temperatures, and at 1300°C at a variety of oxygen fugacities. Transmission electron microscopic and Rutherford backscattering spectroscopic characterization of run products shows that the surface of SiC exposed to IW-2.8 is characterized by a thin ( 1100°C for P^(tot)= 10^(−6) atm and sticking coefficient = 0.01, then the calculated lifetimes would be about 10 yr for 10-μm-diameter grains, essentially independent of temperature. The results thus imply that presolar SiC grains would survive short heating events associated with formation of chondrules (minutes) and calcium-, aluminum-rich inclusions (days), but would have been destroyed by exposure to hot (≥900°C) nebular gases in less than several thousand years unless they were coated with minerals inert to reaction with a nebular gas.

Literature data for major element oxide compositions of most coarse-grained Types A and B inclusions in CV3 chondrites may be in error due to non-representative sampling of spinel relative to other phases because of small sample sizes. When reported compositions are corrected to the solar CaO/Al2O3 ratio by addition or subtraction of spinel, distinct trends result on oxide–oxide plots. These trends lie close to trajectories of bulk compositions of equilibrium condensates calculated for solar or dust-enriched gases under various conditions, except on a plot of MgO vs. SiO2 contents, where there is considerable scatter of the data points to the MgO-poor side of the condensation trajectory. The irreversible process of evaporative mass loss from a liquid droplet into an unsaturated H2 gas is modeled as a series of small equilibrium steps. This model is used to show that evolutionary paths of CMAS liquid compositions are identical for evaporation at all PH2 from 1 × 10−15 to 1 bar, with the ratio of the fraction of the SiO2 evaporated to that for MgO increasing both with increasing temperature from 1700 to 2000 K and with increasing SiO2 content of the starting composition. Such calculations show that compositions of most Type B inclusions can be explained by non-equilibrium evaporation of 10 to 30% of the MgO and 0 to 15% of the SiO2 into an H2 gas at 1700 K from liquid droplets whose compositions originated on any one of many possible equilibrium condensation trajectories. Some Type As may have suffered similar evaporative losses of MgO and SiO2 but at higher temperature. This degree of evaporation is consistent with the amount of Mg and Si isotopic mass fractionation observed in Types A and B inclusions. Evaporation probably happened after most Mg and Si were removed from the nebular gas into lower-temperature condensates.

A combination of hydrodynamic and cross-linking studies were used to investigate self-assembly of the Escherichia coli DNA repair protein UvrB. Though the procession of steps leading to incision of DNA at sites flanking damage requires that UvrB engage in an ordered series of complexes, successively with UvrA, DNA, and UvrC, the potential for self-association had not yet been reported. Gel permeation chromatography, nondenaturing polyacrylamide gel electrophoresis, and chemical cross-linking results combine to show that UvrB stably assembles as a dimer in solution at concentrations in the low micromolar range. Smaller populations of higher order oligomeric species are also observed. Unlike the dimerization of UvrA, an initial step promoted by ATP binding, the monomer-dimer equilibrium for UvrB is unaffected by the presence of ATP. The insensitivity of cross-linking efficiency to a 10-fold variation in salt concentration further suggests that UvrB self-assembly is driven largely by hydrophobic interactions. Self-assembly is significantly weakened by proteolytic removal of the carboxyl terminus of the protein (generating UvrB*), a domain also known to be required for the interaction with UvrC leading to the initial incision of damaged DNA. This suggests that the C terminus may be a multifunctional binding domain, with specificity regulated by protein conformation.

Compact type A (CTA) inclusions are one of the major types of coarse-grained refractory inclusions found in carbonaceous chondrites. They have not been studied in a systematic fashion, leading to some uncertainties and unproven assumptions about their origin. To address this situation, we studied a total of eight CTAs from Allende, Efremovka and Axtell by scanning electron-microscopic and electron and ion-microprobe techniques. These inclusions are very melilite-rich, ranging from ∼60 vol% to nearly monomineralic. Also present are Mg–Al spinel (5–20%), perovskite (trace–∼3%) and, in some samples, Ti-rich (∼17 wt% TiO2tot) fassaite (trace–∼20%), and rhonite (≤1%). Melilite compositions are mostly between Ak15 and Ak40. Chondrite-normalized REE abundance patterns for melilite (flat at ∼10 × CI with positive Eu anomalies) and fassaite (slight HREE enrichment relative to LREE and negative Eu anomalies) are like those for their counterparts in once-molten type B inclusions. The patterns for rhonite have positive slopes from La through Lu and abundances

The small DNA fragment thymidine dinucleotide (pTpT) stimulates photoprotective responses in mammalian cells and intact skin. These responses include increased melanogenesis (tanning) and enhanced repair of DNA damage induced by ultraviolet (UV) light. Here we show that pTpT treatment of human keratinocytes enhances their repair of DNA damaged by the chemical carcinogen benzo(a)pyrene (BP), as determined by increased expression of a transfected BP-damaged reporter plasmid containing the chloramphenicol acetyltransferase (CAT) gene. The pTpT-enhanced repair of this BP-damaged plasmid is accomplished at least in part through activation of the p53 tumor suppressor protein and transcription factor, because p53-null H1299 cells showed enhanced repair only if previously transfected with a p53-expression vector. To elucidate the mechanism of this enhanced DNA repair, we examined the expression of p21 and proliferating cell nuclear antigen (PCNA), proteins known to be regulated by p53, as well as the XPA protein, which is mutated in the inherited repair-deficient disorder xeroderma pigmentosum (XP) group A and is necessary for the recognition of UV-induced DNA photoproducts. The p53, PCNA and XPA proteins were all up-regulated within 48 h after the addition of pTpT. Taken together, these data demonstrate that pTpT-enhanced repair of DNA damaged by either UV irradiation or chemical mutagens can be achieved in human cells by exposure to small DNA fragments at least in part through the activation of p53 and increased expression of p53-regulated genes.

The entire process of nucleotide excision repair (NER) in Escherichia coli has been reconstituted in vitro from purified proteins and defined DNA substrates. However, how this system is organized in vivo in unclear. We report here the isolation and characterization of macromolecular assemblies containing NER and transcription proteins from E. coli. This ensemble consists of at least 17 proteins. They are recruited, as a consequence of DNA damage induced by UV irradiation, to the inner membrane. The UV-induced 6-4 photoproducts are also relocated to the inner membrane following UV-irradiation of the cells. This recruitment process is dependent on the uvrA, uvrC and recA gene products. These results suggest that at least part of the repair process may associate with the inner membrane and also provide insights into understanding the cellular organization of repair processes.

— Through freeze-thaw disaggregation of the Murchison meteorite, we have recovered, in addition to many spinel-hibonite spherules, several hibonite-rich inclusion fragments in which the hibonite has wider ranges in TiO2 contents (e.g., 0.07–8.6 wt% in one inclusion and 2–10 wt% in another) than previously observed within single inclusions. In these inclusions, there are sharp contacts between texturally early, Ti-poor hibonite and relatively late, Ti-rich hibonite, and the two types occur in complex intergrowth textures that are not consistent with crystallization from a melt in a single-stage cooling event. One inclusion has, in addition to relatively TiO2-rich hibonite, some that is virtually TiO2-free but contains ∼1 wt% MgO and ∼1.5 wt% SiO2. Instead of the common substitution of Mg + Ti for 2Al, Mg coupled with Si in this case, probably reflecting crystallization from an unusual, Ti-free silicate liquid. Ion microprobe analyses of Ti-rich and Ti-poor hibonite from this inclusion yield quite similar trace-element patterns and Mg-isotopic compositions. The results are most consistent with formation of Ti-rich hibonite from Ti-poor hibonite by addition of Mg and Ti to the latter by exchange with a hot, Ti-rich liquid. That this occurred without a resolvable change in the Mg-isotopic composition requires that the Ti-rich, second generation of hibonite formed

The Escherichia coli Uvr(A)BC endonuclease (Uvr(A)BC) initiates nucleotide excision repair of a large variety of DNA damages. The damage recognition and incision steps by the Uvr(A)BC is a complex process utilizing an ATP-dependent DNA helix-tracking activity associated with the UvrA2B1 complex. The latter activity leads to the generation of highly positively supercoiled DNA in the presence of E. coli topoisomerase I in vitro. Such highly positively supercoiled DNA, containing ultraviolet irradiation-induced photoproducts (uvDNA), is resistant to the incision by Uvr(A)BC, whereas the negatively supercoiled and relaxed forms of the uvDNA are effectively incised. The E. coli gyrase can contribute to the above reaction by abolishing the accumulation of highly positively supercoiled uvDNA thereby restoring Uvr(A)BC-catalyzed incision. Eukaryotic (calf thymus) topoisomerase I is able to substitute for gyrase in restoring this Uvr(A)BC-mediated incision reaction. The inability of Uvr(A)BC to incise highly positively supercoiled uvDNA results from the failure of the formation of UvrAB-dependent obligatory intermediates associated with the DNA conformational change. In contrast to Uvr(A)BC, the Micrococcus luteus UV endonuclease efficiently incises uvDNA regardless of its topological state. The in vitro topodynamic system proposed in this study may provide a simple model for studying a topological aspect of nucleotide excision repair and its interaction with other DNA topology-related processes in E. coli.

Based on the binding of the UvrAB complex to a promoter region in transcription open complexes (Ahn, B., and Grossman, L. (1996) J. Biol. Chem. 271, 21453-21461) and the requirement of a single-stranded region for UvrAB helicase activity, we examined the binding of UvrAB proteins to synthetic bubble or loop regions in duplex DNA and the role of these regions in translocation of the UvrAB complex as well as incision of DNA damage. We found that the UvrAB complex was able to bind to bubble and loop regions with an affinity similar to that for damaged DNA in the absence of RNAP. The preferential recognition and incision of damaged sites by the UvrAB complex was observed downstream of the bubble or loop region in the strand complementary to the strand along which the UvrAB complex translocates. These results imply that the bubble region generated in duplex DNA by RNAP serves as a preferred entry site for the translocation of the UvrAB complex, and that preferential binding and unidirectional translocation of the UvrAB complex predetermine where incision is to occur.

— Through freeze-thaw disaggregation of the Murchison meteorite, we have recovered a refractory inclusion, HIB-11, that is unique in terms of its texture, mineral compositions, and bulk composition. It consists of anhedral, Y-rich (1.6 wt% Y2O3) perovskite and lathlike spinel grains enclosed in a matrix of fine-grained, Sc-rich (10.5 wt% SC2O3 avg.), Ti-rich (12.6 wt% TiO2 avg., reporting all Ti as TiO2) clinopyroxene. The chondrite-normalized rare earth element (REE) pattern is complex, with light REE (LREE) at ∼10× C1, abundances increasing from Gd through Ho (the latter at ∼104× C1), decreasing through Yb at 200× C1, and Lu at ∼400× C1. The pattern reflects several stages of high-temperature volatility fractionation. Removal of Lu and Er from the source gas in the first condensation event was followed by partial to complete removal of the somewhat less refractory heavy REE, Gd through Ho, in the HIB-11 precursors by condensation from the fractionated residual gas in a second event. Both of these events probably reflect condensation of REE into ZrO2 or a mixed Zr-, Sc-, Ti-, Y-oxide at temperatures too high for hibonite stability. A second, lower-temperature component, which was subsequently added, had fractionated (Nd-poor, Ce-rich) LREE abundances that resulted from condensation from a gas that had undergone prior removal of the more refractory LREE, resulting in enrichment in Ce and the most volatile REE, Eu and Yb. The aggregate was then melted and quickly cooled, forming a fine-grained spherule. This is the first reported inclusion in which the two most refractory REE, Lu and Er, are strongly fractionated from the other REE. An absence of mass fractionation among the Ti isotopes indicates that HIB-11 is not an evaporative residue, implying that volatility fractionation of trace elements took place during condensation. The fact that the two most refractory heavy REE could be separated from the other, only slightly less refractory heavy REE suggests that a wide variety of REE patterns is possible, and that ultrarefractory inclusions with other unusual REE patterns, important recorders of nebular condensation, may yet be discovered.

The Escherichia coli Uvr(A)BC endonuclease acts in a progression of several distinct steps accompanied by changes in the conformation of macromolecular constituents, the overall architecture of the complex, and its stoichiometry. In order to probe these structural changes, we generated monoclonal antibodies (mAbs) to Uvr proteins. The anti-UvrA mAb, A2D1, recognizing the N-terminal zinc-finger region of UvrA, and the anti-UvrB mAb, B2E2, having an epitope within the 43 C-terminal amino acids of UvrB, were purified and further characterized. It was found that A2D1 mAb interacts in solution both with UvrA-UvrB and UvrA-DNA complexes in the presence of the requisite ATP. This implies that the N-terminal zinc-finger of UvrA doesn't play a direct role in its interactions with UvrB and DNA. On the other hand, A2D1 does inhibit formation of the UvrB-damaged DNA preincision complex, apparently by preventing UvrB delivery by UvrA. The interaction of B2E2 with UvrA-UvrB and nucleoprotein complexes, including UvrB, suggests that the highly hydrophobic C-terminal domain of UvrB (i) doesn't participate in its interaction with UvrA, (ii) is accessible to this mAb in an intermediate UvrA-UvrB DNA helix-tracking complex, and (iii) seems to be directly involved in the formation of the preincision complex. These conclusions are supported by the finding that the neutralizing effect of A2D1 and B2E2 on the Uvr(A)BC endonuclease is significantly decreased if the preincision complex is preformed prior to mAbs addition.

A clinic-based case-control study was conducted to determine the association between vitamin supplement use and risk of basal cell carcinoma (BCC) of the skin. The subjects were 131 patients with histopathologically confirmed primary BCC and 200 cancer-free controls with non-premalignant skin disorders. Use of any vitamins (mainly multivitamins and vitamins A, C, and E) was associated with reduced risk of BCC. After controlling for age, sex, cigarette smoking, number of lifetime severe sunburns, and skin actinic elastosis, regular vitamin supplementation was associated with a significantly reduced risk of BCC (odds ratio (OR) = 0.3; 95% confidence interval (CI) = 0.2-0.06). The ORs decreased as the regularity (p < 0.001) and daily doses of supplement used increased, especially for vitamins A (p < 0.005) and E (p < 0.005). Vitamin supplementation was not associated with alterations in cellular DNA repair. These results, however, cannot be considered conclusive because of the relatively low participation rates (131/830 for cases and 200/1406 for controls) due to the requirement of blood donation and more rigorous studies are needed to clarify the effect of supplemental vitamins, particularly of vitamins A and E, on the risk of BCC of the skin.

Expression of a DNA-dependent ATPase activity by the uvrB protein is essential for early steps (preceding incision) in nucleotide excision repair (NER) in E. coli. Yet, in isolation, uvrB lacks any known catalytic ability. Its cryptic ATPase is elicited in NER by association with uvrA, but it can also be turned on by a specific, omp T-mediated proteolytic elimination of the C-terminal 43 amino acids. The truncated protein uvrB{sup *} may serve as a model for the activated structure induced by complex formation with uvrA. To probe the mechanism of activation, which may be expected to require a series of conformational changes, we have introduced the intrinsic fluorophore tryptophan (Trp) into the ATP binding site of uvrB via site-specific mutagenesis.

The cysteine 763 residue in the C-terminal zinc-finger region of Escherichia coli UvrA protein was subjected to random mutagenesis, and the results suggested that the UvrA mutants with a small amino acid (Ser, Ala, or Gly) substituting for the cysteine 763 were almost as active as the wild-type in supporting nucleotide excision repair, but its replacement with a large, bulky amino acid (Tyr, Trp, or Phe) rendered the mutants inactive. The C763F mutant UvrA protein was purified for further characterization, and it was found this mutant UvrA protein lost its DNA binding (single-stranded or double-stranded DNA) activity and those other activities dependent on DNA binding, such as formation of damage-specific UvrA2B complexes and the supercoiling reaction. However, this mutant protein retained vigorous ATPase activity and was capable of negatively complementing the wild-type UvrA in JM109 strain. The purified C763F mutant UvrA protein contains a single zinc ion/molecule, half that of the wild-type. It appears that the C763F mutation destabilizes the zinc-anchored structure in the C-terminal zinc finger region, and as a result, the C763F mutant UvrA protein lost its ability to bind DNA.

Watson, which was found in 1972 in South Australia, contains the largest single silicate rock mass seen in any known iron meteorite. A comprehensive study has been completed on this unusual meteorite: petrography, metallography, analyses of the silicate inclusion (whole rock chemical analysis, INAA, RNAA, noble gases, and oxygen isotope analysis) and mineral compositions (by electron microprobe and ion microprobe). The whole rock has a composition of an H-chondrite minus the normal H-group metal and troilite content. The oxygen isotope composition is that of the silicates in the IIE iron meteorites and lies along an oxygen isotope fractionation line with the H-group chondrites. Trace elements in the metal confirm Watson is a new IIE iron. Whole rock Watson silicate shows an enrichment in K and P (each approximately 2X H-chondrites). The silicate inclusion has a highly equilibrated igneous (peridotite-like) texture with olivine largely poikilitic within low-Ca pyroxene: olivine (Fa20), opx (Fs17Wo3), capx (Fs9Wo14)(with very fine exsolution lamellae), antiperthite feldspar (An1-3Or5) with less than 1 micron exsolution lamellae (An1-3Or greater than 40), shocked feldspar with altered stoichiometry, minor whitlockite (also a poorly characterized interstitial phosphate-rich phase) and chromite, and only traces of metal and troilite. The individual silicate minerals have normal chondritic REE patterns, but whitlockite has a remarkable REE pattern. It is very enriched in light REE (La is 720X C1, and Lu is 90X C1, as opposed to usual chonditic values of approximately 300X and 100-150X, respectively) with a negative Eu anomaly. The enrichment of whole rock K is expressed both in an unusually high mean modal Or content of the feldspar, Or13, and in the presence of antiperthite.

Most spinel grains in Murchison acid residues are Mg-, Al-rich, 16O-rich (δ18O = −50%.), small (10–30 μm) and probably from refractory inclusions. They are quite unlike spinels we have recovered from Murchison by freeze-thaw disaggregation, density separation, and handpicking. As reported here, the latter spinels contain up to 37 wt% Cr2O3 and up to 17 wt% FeO, are not 16O-enriched (δ18O = 1.9 ± 2.4%.), are coarse (60–325 μm), and are not from refractory inclusions. From backscattered electron images of fifty-seven such grains, we recognize five zoning types defined by variations in Cr2O3 contents: patchy (56%); homogeneous (21%); chevron (10.5%); gradational (9%); and core-rim (3.5%). Many grains have silicate inclusions, the most common being small, anhedral grains of diopside with 12–24 wt% Al2O3 and up to 3.7 wt% TiO2. Eleven spinel samples occur with forsteritic (Fo95–99) olivine; in most cases, the spinel partially encloses the olivine. Cr-bearing spinel was found in situ in two Al-rich chondrules (one with homogeneous spinel, the other with homogeneous, gradational and core-rim spinel, and both with forsterite and aluminous diopside); in two irregularly shaped, olivine-bearing inclusions (one with homogeneous spinel, the other patchy); and attached to an isolated olivine grain (patchy). Observation of homogeneous, gradational, and core-rim type spinels in chondrules and basalts shows that grains with these zoning patterns can crystallize from liquids, although, in Murchison, chondrules with the appropriate compositions and sufficiently coarse textures to yield the separated spinels are exceedingly rare. Chevronzoned grains also could have formed in chondrules; alternatively, they may have acquired their oscillatory zoning patterns by cycling through different P-T-ƒ O 2 regimes in the solar nebula during their formation. The patchy spinel grains were probably never molten and they most likely formed by sintering of aggregates of smaller spinel grains which were enriched in Cr and Fe to varying degrees. In spite of their various crystallization and thermal histories, the spinels all have normal oxygen and chromium isotopic compositions, consistent with formation from a single, well-mixed nebular reservoir. Based on the known slow rates of diffusion of oxygen in MgO, Al2O3, and MgAl2O4, it is unlikely that the spinels of this study formed from an isotopically anomalous reservoir and later re-equilibrated with a normal one; it is more likely that they have retained their original isotopic compositions. We see no evidence for anomalous Cr, which had been reported by others.

The biochemical properties of the Escherichia coli UvrA tandem ATPase site mutants in nucleotide excision repair have been studied. In these and earlier studies it was found that ATP binding is required for protein-protein and nucleoprotein association reactions, whereas the dissociation reactions are driven by the hydrolysis of ATP. The self-association of UvrA to form the reactive dimeric species UvrA2 is driven by nucleotide binding, but its dissociation from DNA requires ATP hydrolysis. Similarly, ATP binding drives those allosteric changes in DNA topology during UvrA2-nucleoprotein formation (Oh, E.Y., and Grossman, L. (1986) Nucleic Acids Res. 14, 8557-8571). The manifestation of the UvrB-associated cryptic ATPase requires UvrA and DNA in a helicase-catalyzed supercoiling reaction. The UvrA2B helicase activity requires ATP hydrolysis by the C-terminal ATPase site of UvrA in addition to UvrB. ATP hydrolysis by the C-terminal ATPase site of UvrA also participates in the localization of damaged sites contributing to the formation of damage-specific high affinity nucleoprotein complexes. The levels of complementation to UV survival by the ATPase site mutants of UvrA (Thiagalingam, S., and Grossman, L. (1991) J. Biol. Chem. 266, 11395-11403) correspond to its ability to self-bind and translocate in combination with the UvrB subunit in its search for damaged sites during the preincision mode of nucleotide excision.

The repair by UvrABC endonuclease of two major adducts formed by aflatoxin B1 in DNA was found to be similar. Aflatoxin epoxide was used to generate the aflatoxin B1.N7-guanine adduct which can convert to aflatoxin B1-formamidopyrimidine adduct. The reaction of the aflatoxin B1 epoxide with DNA follows pseudo-first order kinetics. The DNA sequence-specific relative reactivity of the epoxide is the same as previously observed for aflatoxin B1 activated by liver microsomes, therefore strongly reinforcing the notion that aflatoxin B1 reacts with DNA through the epoxide intermediate. For the majority of lesion sites, a high affinity protein-DNA complex was formed from the UvrA and the UvrB proteins with similar efficiency to both adducts, and to pyrimidine dimers, and then nicks the DNA when UvrC was added. The two incisions are at the eighth phosphodiester moiety 5' and the sixth phosphodiester moiety 3' of a modified guanine nucleotide. Both incisions appeared to be concerted. For some sites, the DNA sequence can alter the relative incision efficiency up to 15-fold. However, the majority of these AFB1 lesion structures in most DNA sequences are similar with respect to recognition by this nucleotide excision repair enzyme. Therefore the observation that the aflatoxin B1.N7-guanine lesion is removed rapidly, while the aflatoxin B1-formamidopyrimidine lesion persists in the mammalian cell may have other mechanistic explanations.

The functions of each of the three subunits of the damage-specific UvrABC endonuclease is currently being studied by systematically mutagenizing the corresponding genes to generate mutant proteins for characterization in vitro. In this communication, we describe the construction of C-terminal deletion mutants of the UvrA protein and a procedure to purify the mutant and wild-type UvrA proteins from inclusion bodies in cells overexpressing the recombinant proteins. The method yields highly purified proteins with between 10 and 50% of the specific activity of wild-type UvrA purified by conventional techniques from the soluble fraction. The wild-type UvrA protein purified by this method had the properties of significant and selective loss of activity in assays of incision of damaged DNA, while still retaining high levels of the other unique molecular phenotypic properties associated with intact UvrA. Furthermore, the demonstration of the absolute requirement for zinc during refolding for recovery of activity is the first evidence that the zinc previously shown to be associated with the UvrA protein is in fact a necessary component for its function.

The UvrA protein is the DNA binding and damage recognition subunit of the damage-specific UvrABC endonuclease. In addition, it is an ATPase/GTPase, and the binding energy of ATP is linked to dimerization of the UvrA protein. Furthermore, the UvrA protein interacts with the UvrB protein to modulate its activities, both in solution and in association with DNA, where the UvrAB complex possesses a helicase activity. The domains of the UvrA protein that sponsor each of these activities were localized within the protein by studying the in vitro properties of a set of purified deletion mutants of the UvrA protein. A region located within the first 230 amino acids was found to contain the minimal region necessary for interactions with UvrB, the UvrA dimerization interface was localized to within the first 680 amino acids, and the DNA binding domain lies within the first 900 amino acids of the 940-amino acid UvrA protein. Two damage recognition domains were detected. The first domain, which coincides with the DNA binding region, is required to detect the damage. The second domain, located on or near the C-terminal 40 amino acids, stabilizes the protein-DNA complex when damage is encountered.

The roles of the two tandemly arranged putative ATP binding sites of Escherichia coli UvrA in UvrABC endonuclease-mediated excision repair were analyzed by site-directed mutagenesis and biochemical characterization of the representative mutant proteins. Evidence is presented that UvrA has two functional ATPase sites which coincide with the putative ATP binding motifs predicted from its amino acid sequence. The individual ATPase sites can independently hydrolyze ATP. The C-terminal ATPase site has a higher affinity for ATP than the N-terminal site. The invariable lysine residues at the ends of the glycine-rich loops of the consensus Walker type A motifs are indispensable for ATP hydrolysis. However, the mutations at these lysine residues do not significantly affect ATP binding. UvrA, with bound ATP, forms the most favored conformation for DNA binding. The initial binding of UvrA to DNA is chiefly at the undamaged sites. In contrast to the wild type UvrA, the ATPase site mutants bind equally to damaged and undamaged sites. Dissociation of tightly bound nucleoprotein complexes from the undamaged sites requires hydrolysis of ATP by the C-terminal ATPase site of UvrA. Thus, both ATP binding and hydrolysis are required for the damage recognition step enabling UvrA to discriminate between damaged and undamagedmore » sites on DNA.« less

T-21 silica, a commonly used encapsulation material in neutron activation analysis of small samples, was analyzed by INAA to determine trace levels of the following impurities: Na, Sc, Cr, Fe, Co, Ni, Zn, Br, Sb, La, Ce, Sm, Tb, Hf and Au. In the unprocessed form supplied by the manufacturer, pieces of T-21 weighing 50 mg contain ∼6·10−2 μg Na; ∼9·10−3 μg Fe; ∼3·10−7–7·10−6 μg light REE, Sc and Hf and ∼4·10−8 μg Tb and Au. In a series of glass-blowing steps, in which unfiltered gases were used for fuel, and cleaning steps, in which reagent grade aqua regia was used, irradiation vials were produced which contain higher average levels of Sc, La, Sm, Tb and Hf, by factors ranging from 1.3 for Sm to 11.5 for La, and lower average levels of Co, Na and Fe, by factors of 1.4, 2.0 and 4.0, respectively, than the unprocessed material. These contamination levels lead to blank corrections of 21% for La, 2.6% for Ce and 3.0% for Hf in 40 μg samples of refractory inclusions from carbonaceous chondrites, if counted in their irradiation vials.

The role of UvrB in determining the nucleotide dependence of Escherichia coli excision repair has been investigated. The mutation of lysine 45 in the ATPase motif of UvrB to alanine leads to an acute defect in ATP hydrolysis and failure to support incision of UV-damaged DNA. This ATP hydrolysis activity is not required for interaction of UvrB with UvrA in solution, or for formation of a damage-independent nucleoprotein complex in the presence of UvrA and nucleotide. This UvrB mutant fails, however, to support damage-specific nucleoprotein complex formation, and does not participate in a UvrA-UvrB-dependent helicase-like activity. We conclude from these results that mutation at lysine 45 in the ATPase motif of UvrB specifically inhibits a key step in nucleotide excision repair involving the UvrB ATPase-dependent translocation of nucleoprotein complexes from undamaged to damaged DNA sites.

A newly developed cell-free system was used to study DNA repair synthesis carried out by extracts from human cell lines in vitro. Extracts from a normal human lymphoid cell line and from cell lines established from individuals with hereditary dysplastic nevus syndrome perform damage-dependent repair synthesis in plasmid DNA treated with cis- or trans-diamminedichloro-platinum(II) or irradiated with ultraviolet light. Cell extracts of xeroderma pigmentosum origin (complementation groups A, C, D, and G) are deficient in DNA repair synthesis. When damaged plasmid DNA was pretreated with purified Escherichia coli UvrABC proteins, xeroderma pigmentosum cell extracts were able to carry out DNA repair synthesis. The ability of E. coli UvrABC proteins to complement xeroderma pigmentosum cell extracts indicates that the extracts are deficient in incision, but can carry out later steps of repair. Thus the in vitro system provides results that are in agreement with the incision defect found from studies of xeroderma pigmentosum cells.

Structural intermediates generated during incision of damaged DNA by the Uvr(A)BC endonuclease were probed with monoclonal antibodies (mAbs) raised against the Escherichia coli UvrB protein. It was found that the epitope of B2C5 mAb, mapped at amino acids (aa) 171–278 of UvrB, is not accessible in any of the preformed Uvr intermediates. Preformed B2C5-UvrB immunocomplexes, however, inhibited formation of those intermediates. B2C5 mAb seems to interfere with the formation of the UvrA-UvrB complex due to overlapping of its epitope and the UvrA binding region of UvrB. Conversely, the epitope of B3C1 mAb (aa 1–7 and/or 62–170) was accessible in all Uvr intermediates. The epitope of B*2E3 mAb (aa 171–278) was not accessible in any of the nucleoprotein intermediates preceding UvrB-DNA preincision complex. However, B*2E3 was able to immunoprecipitate this complex and to inhibit overall incision. B2A1 mAb (aa 8–61) inhibited formation of those Uvr intermediates requiring ATP binding and/or hydrolysis by UvrB. B*2B9 mAb (aa 473–630) inhibited Uvr nucleoprotein complexes involving UvrB. B*2B9 seems to prevent the binding of the UvrA-UvrB complex to DNA. The epitope of the B*3E11 mAb (aa 379–472) was not accessible in Uvr complexes formed at damaged sites. These results are discussed in terms of structure-functional mapping of UvrB protein.

The DNA-dependent ATPase activity of UvrB is required to support preincision steps in nucleotide excision repair in Escherichia coli. This activity is, however, cryptic. Elicited in nucleotide excision repair by association with the UvrA protein, it may also be unmasked by a specific proteolysis eliminating the C-terminal domain of UvrB (generating UvrB*). We introduced fluorescent reporter groups (tryptophan replacing Phe47 or Asn51) into the ATP binding motif of UvrB, without significant alteration of behavior, to study both nucleotide binding and those conformational changes expected to be essential to function. The inserted tryptophans occupy moderately hydrophobic, although potentially heterogeneous, environments as evidenced by fluorescence emission and time-resolved decay characteristics, yet are accessible to the diffusible quencher acrylamide. Activation, via specific proteolysis, is accompanied by conformational change at the ATP binding site, with multiple changes in emission spectra and a greater shielding of the tryptophans from diffusible quencher. Titration of tryptophan fluorescence with ATP has revealed that, although catalytically incompetent, UvrB can bind ATP and bind with an affinity equal to that of the active UvrB* form (Kd of approximately 1 mM). The ATP binding site of UvrB is therefore functional and accessible, suggesting that conformational change either brings amino acid residues into proper alignment for catalysis and/or enables response to effector DNA.

Monoclonal antibodies (mAbs) were generated that recognize UvrA and UvrB proteins. These proteins are components of the Uvr(A)BC endonuclease, which initiates nucleotide excision repair in Escherichia coli. mAbs, which can be used for probing of structural intermediates of Uvr(A)BC endonuclease functioning, were selected for their ability to: (i) recognize different epitopes; (ii) have a high-affinity for native antigenic protein; (iii) preserve functionality of the Uvr protein in immunocomplex. The adherence of anti-Uvr mAbs with these criteria was verified by additivity and competition tests, and by their influence on the ATPase activities of UvrA and UvrB*, the functionally active proteolytic fragment of UvrB. Two out of twelve anti-UvrA and seven out of thirteen anti-UvrB/anti-UvrB* hybridoma lines were shown to satisfy these criteria. Recognition of UvrA and UvrB deletion mutant proteins by mAbs was used to map their epitopes. Epitopes of A2D1 and A2B1 mAbs were mapped to regions of amino acids 230–281 and 560–680 of UvrA, respectively. Epitopes of anti-UvrB/UvrB* mAbs were assigned to the following amino acid regions of UvrB: B2A1, 8–61; B2C5 and B*2E3, 171–278; B2E2, 631–673; B3C1, 1–7 and/or 62–170; B*2B9, 473–630; B*3E11, 379–472. The ability of selected mAbs to neutralize the incision function of Uvr(A)BC was analyzed. The results are discussed in terms of the applicability of these mAbs to probe the structures of intermediates in the functioning of Uvr(A)BC.

One form of nucleotide excision repair (NER) is known to be functionally coupled to transcription, but the nature of this functional link in Escherichia coli is still unclear. Here we have employed the isolated membrane-associated nucleoids from E.coli to examine this issue. We show that the isolated nucleoid fraction is capable of excision of UV-induced pyrimidine dimers when reconstituted with a cytoplasmic fraction resolved by sucrose gradient fractionation. This excision activity by UvrABC is sensitive to rifampicin and is dependent on transcription. By using crosslinking and immunoprecipitation, the damage recognition protein, UvrA, was found to be specifically associated with the RNA polymerase beta subunit on the chromosomal DNA independent of DNA damage. It suggests that at least in one of the NER pathways the search for damage may be directly linked to RNA polymerase. In addition, the role of transcription in the unfolding of the nucleoid structure to allow repair enzymes to gain access to the damaged DNA is described. This study provides insight into the understanding of the transcription-repair coupling in vivo.

Aging in humans carries an increased risk of skin cancer, a disorder linked to somatic mutations in sun damaged skin. DNA repair plays a major role in protection against sun damage. We found an age-related decline in post-UV DNA repair capacity (measured by the ability to repair a UV-treated plasmid (pCMVcat)) of-0.6% per year (p = 0.0001) in cultured primary skin fibroblasts from normal donors from the first to the tenth decade of life. There was a corresponding age-related increase in post-UV mutability (measured as mutations introduced into a transfected, UV-treated plasmid (pSP189)) of +0.6% per year (p = 0.001) in lymphoblastoid cell lines from normal donors of the same age range. This study indicates that aging in humans is associated with decreasing ability to process new UV-induced DNA damage and this age-related reduction in DNA repair capacity and increase in DNA mutability is reflected in cultured skin and blood cells.

Transcription when coupled to nucleotide excision repair specifies the location in active genes where preferential DNA repair is to take place. During DNA damage-induced recruitment of RNA polymerase (RNAP), there is a physical association of the beta subunit of Escherichia coli RNAP and the UvrA component of the repair apparatus (G. C. Lin and L. Grossman, submitted for publication). This molecular affinity is reflected in the ability of the RNAP to increase, in a promoter-dependent manner, DNA supercoiling by the UvrAB complex. In the presence of the RNAP, the UvrAB complex is able to bind to promoter regions and to translocate in a 5' to 3' direction along the non-transcribed strand. As a consequence of this helicase-catalyzed translocation, preferential incision of DNA damaged sites occurs downstream on the transcribed strand. Because of the helicase directionality, the initial binding of the UvrAB complex to the transcribed strand would inevitably lead to its collision with the RNAP. These results imply that the RNAP-induced DNA structure in the vicinity of the transcription start site signals a landing or entry site for the UvrAB complex on DNA.

The Escherichia coli UvrA protein possesses a stretch of amino acids, 494 to 513, that matches the consensus sequence of the helix-turn-helix motif of many sequence-specific DNA binding proteins. It also has two zinc finger motif regions and two ATP binding sites. To study the potential roles of both helix-turn-helix and zinc finger motifs in the functioning of UvrA protein, random mutations were created in these motif regions by degenerate oligonucleotide-directed mutagenesis. Using this method, 12 single substitution mutants (eight in the helix-turn-helix motif region, one in the N-terminal zinc finger region, and three in the C-terminal zinc finger region) were isolated that failed to confer UV resistance in the E. coli strain deleted of the uvrA gene. One "hyper" UV-resistant mutant, G275A, was identified that conferred significantly more UV resistance than the wild type in the MH1-delta A strain. To further investigate the mechanism of failure of these mutant UvrA proteins to support nucleotide excision repair, two mutant UvrA proteins, G502D and V508D, were selected for purification and characterization, since they carry mutations at the positions offered as the putative constellation for the helix-turn-helix motif. The binding affinity of these two mutants for nonirradiated plasmid DNA was unaffected by the mutations. Both mutant proteins exhibited substantial ATPase activity, and together with the UvrB protein, they were capable of generating positively supercoiled plasmid DNA from the relaxed form in the presence of ATP and bacterial topoisomerase I. However, both mutant proteins failed to respond to UV damage in the filter binding assay and were incapable of forming 2 x SSC-resistant nucleoprotein complexes with UvrB protein on UV-irradiated plasmid DNA. Taking these properties together, it appears that the mutations in the helix-turn-helix motif region impaired the UvrA protein's ability to recognize UV damage, while its other activities were largely unaffected. Interestingly, ERCC-3, a human DNA repair protein, also has a similar helix-turn-helix motif. Given the highly conserved nature of repair proteins in general, this observation raises the possibility that both procaryotes and eucaryotes might use similar mechanisms to recognize damaged sites in their genomes.

The initial stages in the repair of damaged DNA by the Escherichia coli uvr system involve the recognition of damage by UvrA. We have examined in detail the binding of UvrA to DNA randomly damaged by ultraviolet light, undamaged DNA, and single-stranded DNA using nitrocellulose filter binding and gel mobility shift assays to arrive at the following model: UvrA dimers bind specifically to damaged DNA both in the presence and in the absence of ATP. The dimerization of UvrA is promoted by UvrA concentrations greater than 1 nM, the presence of ATP, or physiological temperatures, and the dimerization step dominates the temperature dependence of UvrA binding to DNA damaged by ultraviolet light. The apparent association constant for specific binding is dependent on the concentration of UvrA due to coupled dimerization, aggregation, and nonspecific binding reactions. At 1 nM UvrA, either with or without ATP, Kuv approximately 10(9) M-1. The binding of UvrA to undamaged DNA is 10(3)-10(4)-fold weaker than the damage-specific binding. Both the strength of damage-specific binding and the discrimination between damaged and undamaged sites are affected by the salt concentration. The kinetics of association and dissociation reactions indicate that the primary effects of ATP are on the extent of UvrA dimerization rather than on the properties of the UvrA-uvDNA complex. The complexity of the interaction of UvrA, ATP, and DNA is indicated by the opposing effects of ATP binding and hydrolysis on UvrA dimerization.

It is the thesis of this paper that DNA can be utilized as a target to detect environmental agents that can modify DNA directly or indirectly through metabolic activation. Expression of DNA modification will be achieved by using as a target a bacterial gene encoding an easily assessable enzyme. The modified gene will be physically introduced into DNA repair deficient lymphoid lines. The level of damage will be reflected as loss of gene activity manifested as reduction of the specific enzyme activity. This extremely sensitive and specific assay, in its essence, is a “Host Cell Reactivation” (HCR) in which damaged viruses are repaired by their hosts.

Nucleotide excision is initiated by the UvrABC endonuclease system in which the initial DNA interaction is with UvrA which was dimerized in the presence of ATP. Nucleoprotein formation most likely takes place on undamaged regions of DNA by (UvrA)2 which has been dimerized in the presence of ATP. Topological unwinding of DNA, driven by ATP binding, is increased by the presence of UvrB to approximately a single helical turn. The Uvr(A)2B complex translocates to a damaged site by the combined Uvr(A)2B helicase in which the driving force is provided by the UvrB-associated ATPase. The dual incision reaction is initiated by the binding of the UvrC protein to the Uvr(A)2B-nucleoprotein complex. The proteins in this post-incision nucleoprotein complex do not turn over and require the presence of the UvrD protein and DNA polymerase I under polymerizing conditions. The final integrity of the DNA strands is restored with polynucleotide ligase.

A detailed mineralogical study of HAL was initiated to elucidate the origin of this inclusion because LEE et al. (1979) had found large mass fractionation effects and small nuclear effects in its Ca isotopic composition, but no ^(26)Mg excesses in samples of it with very high ^(27)Al/^(24)Mg ratios. HAL's 1.0 mm radius interior consists almost entirely of three hibonite crystals and is surrounded by a 2.0 mm thick, multi-layered rim. The first layer, called the black rim, is black and compact, resembles a devitrified glass and contains an anisotropic Al-Fe-oxide similar to hercynite in composition. This is followed by a friable rim sequence, layer I of which is predominantly feldspathoids with minor anorthite, Ti-Fe-oxide and Al-Fe-oxide. Layer II contains abundant perovskite, plus grossular, andradite and pyroxene in addition to the minerals of layer I. Layer III is mostly Ca-phosphate, possibly hydroxy-apatite and perovskite. Layer IV is rich in hibonite, Al-Fe-oxide, perovskite, nepheline and the two garnets, lacks Ca-phosphate but contains traces of a Ti-Sc-Zr-oxide. Layer V is rich in Al-Fe-oxide, pyroxene, nepheline, the two garnets and olivine whose crystals display peculiar rectangular cross-sections. The black rim does not completely surround the hibonite core. Sectors of the friable rim exist where layer I is missing and where the mineralogy of adjacent layers is no different from that of the same layers in other sectors. Pentlandite, nickel-iron and barrel-shaped olivine crystals, minerals typical of the matrix of Allende and found nowhere else in HAL, are found in layer V and increase in abundance toward its exterior, as if grains of these phases accreted together with the other minerals of layer V. This layer also contains alternating olivine-rich and garnet-, pyroxene-rich bands, resembling rhythmic layering. For these reasons, we conclude that each of the layers of the friable rim formed by the accretion of an assemblage of condensate grains rather than by complete reaction of a precursor to HAL with a nebular gas. Thus, the unusual isotopic characteristics of HAL are thought to have been inherited from a nebular reservoir which was isotopically distinct from that which gave rise to the bulk of the material in Allende. HAL's mineralogical peculiarities indicate that its formation reservoir was also chemically distinct from the latter one.

Greyish-brown irregularly-shaped aggregates composed predominantly of olivine make up nearly 2% of the Allende meteorite by volume. Many of the aggregates are constructed of subspherical lumps of micron-sized crystals of olivine, pyroxene, nepheline and sodalite surrounded by coarser-grained olivine. Rarely, anorthite, spinel and perovskite are also present. The olivine ranges in composition from Fo64 to Fo99. Pyroxenes range from aluminous diopside to hedenbergite to very Al-rich and Ti-Al-rich varieties. The nepheline contains 1.6-2.4% K2O and 1.6-5.2% CaO but the sodalite is significantly poorer in these elements. The spinel contains 2.1-13.4% FeO. Textural information and oxygen isotopic data suggest that the aggregates are composed of primary, solid condensates from the solar nebula. The perovskite, spinel and Ti-Al-rich pyroxenes are the remains of high-temperature condensates, but the olivine compositions and the presence of feldspathoids indicate that some of the grains continued to react with the solar nebular vapor in the temperature range 500-900 K.

We have analyzed the ATPase activity exhibited by the UvrABC DNA repair complex. The UvrA protein is an ATPase whose lack of DNA dependence may be related to the ATP induced monomer-dimer transitions. ATP induced dimerization may be responsible for the enhanced DNA binding activity observed in the presence of ATP. Although the UvrA ATPase is not stimulated by dsDNA, such DNA can modulate the UvrA ATPase activity by decreases in Km and Vm and alterations in the Ki for ADP and ATP-gamma-S. The induction of such changes upon binding to DNA may be necessary for cooperative interactions of UvrA with UvrB that result in a DNA stimulated ATPase for the UvrAB protein complex. The UvrAB ATPase displays unique kinetic profiles that are dependent on the structure of the DNA effector. These kinetic changes correlate with changes in footprinting patterns, the stabilization of protein complexes on DNA damage and with the expression of helicase activity.

Enthalpies of formation were determined for two calcium aluminate phases, CaAl4O7 and CaAl12O19, using high-temperature alkali borate solution calorimetry. The aluminates were synthesized by multiple-cycle heating and grinding stoichiometric mixtures of CaCO3 and Al2O3, and the products were characteized by X-ray diffraction and SEM microbeam analysis. The data on impurities (CaAl4O7 was found to be about 89.00 percent pure by weight and the CaAl12O19 samples about 91.48 percent pure) were used to correct the heat of solution values of the synthetic products. The enthalpies of formation, at 1063 K, from oxides, were found to be equal to -(25.6 + or - 4.7) kJ/g.f.w. for CaAl4O7 and -(33.0 + or - 9.7) kJ/g.f.w. for CaAl12O19; the respective standard enthalpies of formation from elements, at 298 K, were estimated to be -4007 + or - 5.2 kJ/g.f.w. and -10,722 + or - 12 kJ/g.f.w.

UvrB is specifically proteolyzed in Escherichia coli cell extracts to UvrB*. UvrB* is capable of interacting with UvrA in an apparently similar manner to the UvrB, however UvrB* is defective in the DNA strand displacement activity normally displayed by UvrAB. Whereas the binding of UvrC to a UvrAB-DNA complex leads to DNA incision and persistence of a stable post-incision protein-DNA complex, the binding of UvrC to UvrAB* leads to dissociation of the protein complex and no DNA incision is seen. The factor which stimulates this proteolysis has been partially purified and its substrate specificity has been examined. The protease factor is induced by "stress" and is under control of the htpR gene. The potential role of this proteolysis in the regulation of levels of active repair enzymes in the cell is discussed.

The cloned uvrA and uvrB genes of Escherichia coli K-12 were amplified by linkage to the PL promoter of plasmid pKC30. The uvrC gene was amplified in the high-copy-number plasmid pRLM 24. The three gene products (purified in each case to greater than 95% purity) and ATP are required to effectively incise UV-damaged DNAs. The uvrABC proteins bind tightly to damaged sites in DNA, requiring the initial attachment of the uvrA protein in the presence of ATP before productive binding of the uvrB and uvrC proteins. Using a cloned tandem double insert of the lac p-o region as a damaged DNA substrate for the uvrABC complex and analyzing the incision both 5' and 3' to each pyrimidine dimer, we found that one break occurs 7 nucleotides 5' to a pyrimidine dimer and a second break is made 3-4 nucleotides 3' from the same pair of pyrimidines in the dimer. No such breaks are found in the strand complementary to the dimer. The size of the incised fragment in the DNA suggests that incision may be coordinated with excision reactions in repair processes.