Human DNA ligase and DNA polymerase as molecular targets for heavy metals and anticancer drugs

DNA ligase and DNA polymerase play important roles in DNA replication, repair, and recombination. Frequencies of spontaneous and chemical- and physical-induced mutations are correlated to the fidelity of DNA replication. This dissertation elucidates the mechanisms of the DNA ligation reaction by DNA ligases and demonstrates that human DNA ligase I and DNA polymerase $\alpha$ are the molecular targets for two metal ions, Zn$\sp{2+}$ and Cd$\sp{2+},$ and an anticancer drug, F-ara-ATP.^ Human DNA ligases were purified to homogeneity and their AMP binding domains were mapped. Although their AMP-binding domains are similar, there could be difference between the two ligases in their DNA binding domains.^ The formation of the AMP-DNA intermediate and the successive ligation reaction by human DNA ligases were analyzed. Both reactions showed their substrate specificity for ligases I and II, required Mg2+, and were inhibited by ATP.^ A protein inhibitor from HeLa cells and specific for human DNA ligase I but not ligase II and T4 ligase was discovered. It reversibly inhibited DNA ligation activity but not the AMP-binding activity due to the formation of a reversible ligase I-inhibitor complex.^ F-ara-ATP inhibited human DNA ligase I activity by competing with ATP for the AMP-binding site of DNA ligase I, forming a ligase I-F-ara-AMP complex, as well as when it was incorporated at 3$\sp\prime$-terminus of DNA nick by DNA polymerase $\alpha.$^ All steps of the DNA ligation reaction were inhibited by Zn$\sp{2+}$ and Cd$\sp{2+}$ in a concentration-dependent manner. Both ions did not show the ability to change the fidelity of DNA ligation reaction catalyzed by human DNA ligase I. However, Zn$\sp{2+}$ and Cd$\sp{2+}$ showed their contradictory effects on the fidelity of the reaction by human DNA polymerase $\alpha.$ Zn$\sp{2+}$ decreased the frequency of misinsertion but less affected that of mispair extension. On the contrary, Cd$\sp{2+}$ increased the frequencies of both misinsertion and mispair extension at very low concentration. Our data provided strong evidence in the molecular mechanisms for the mutagenicity of zinc and cadmium, and were comparable with the results previously reported. ^

Metabolism and actions of 2 -chloro-2$\sp\prime$-fluoro-arabinosyladenine

2-Chloro-9-(2-deoxy-2-fluoro-$\beta $-D-arabinofuranosyl)adenine(Cl-F-ara-A) is a new deoxyadenosine analogue which is resistant to phosphorolytic cleavage and deamination, and exhibits therapeutic activity for both leukemia and solid tumors in experimental systems. To characterize its mechanism of cytotoxicity, the present study investigated the cellular pharmacology and the biochemical and molecular mechanisms of action of Cl-F-ara-A, from entrance of the drug into the cell, chemical changes to active metabolites, targeting on different cellular enzymes, to final programmed cell death response to the drug treatment.^ Cl-F-ara-A exhibited potent inhibitory action on DNA synthesis in a concentration-dependent and irreversible manner. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, and their elimination was non-linear with a prolonged terminal phase, which resulted in prolonged dNTP depression. Ribonucleotide reductase activity was inversely correlated with the cellular Cl-F-ara-ATP level, and the inhibition of the reductase was saturated at higher cellular Cl-F-ara-ATP concentrations. The sustained inhibition of ribonucleotide reductase and the consequent depletion of deoxynucleotide triphosphate pools result in a cellular Cl-F-ara-ATP to dATP ratio which favors analogue incorporation into DNA.^ Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA. A much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA, suggesting that the incorporation of Cl-F-ara-A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A.^ Cl-F-ara-ATP competed with dATP for incorporation into the A-site of the extending DNA strand catalyzed by both DNA polymerase $\alpha$ and $\varepsilon$. The incorporation of Cl-F-ara-AMP into DNA resulted in termination of DNA strand elongation, with the most pronounced effect being observed at Cl-F-ara-ATP:dATP ratio $>$1. The presence of Cl-F-ara-AMP at the 3$\sp\prime$-terminus of DNA also resulted in an increased incidence of nucleotide misincorporation in the following nucleotide position. The DNA termination and the nucleotide misincorporation induced by the incorporation of Cl-F-ara-AMP into DNA may contribute to the cytotoxicity of Cl-F-ara-A. ^

2-methylthio-N$\sp6$-dimethylallyl modification of adenosine 37 of tRNAs in {\it Escherichia coli\/} K-12

The hypermodified, hydrophobic 2-methylthio-N$\sp6$-(dimethylallyl)-adenosine (ms${2{\cdot}6}\atop1$A) residue occurs $3\sp\prime$ to the anticodon in tRNA species that read codons beginning with U. The first step (i$\sp6$A37 formation) of this modification is catalyzed by dimethylallyl diphosphate:tRNA dimethyallyltransferase (EC 2.5.1.8), which is the product of the miaA gene. Subsequent steps were proposed to be catalyzed by MiaB and MiaC enzymes to complete the ms${2{\cdot}6}\atop1$A37 modification. The study of functions of the ms${2{\cdot}6}\atop1$A37 is very important because this modified base is one of the best candidates for a role in global control in response to environmental stress. This dissertation describes the further delineation of functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli K-12 cells. This work provides significant information on functions of tRNA modifications in E. coli cells to adapt to stressful environmental conditions. Three hypotheses were tested in this work.^ The first hypothesis tested was that non-optimal translation processes cause increased spontaneous mutagenesis by the induction of SOS response in starving cells. To test this hypothesis, I measured spontaneous mutation rates of wild type cells and various mutant strains which are defective in tRNA modification, SOS response, or oxidative damage repair. I found that the miaA mutation acts as a mutator that increased Lac$\sp+$ reversion rates and Trp$\sp+$ reversion frequencies of the wild-type cells in starving conditions. However, the lexA3(Ind)(which abolishes the induction of SOS response) mutation abolished the mutator phenotype of the miaA mutant. The recA430 mutation, not other identified SOS genes, decreased the Lac$\sp+$ reversion to a less extent than that of the lexA3(Ind) mutation. These results suggest that RecA together with another unidentified SOS gene product are responsible for the process.^ The second hypothesis tested was that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ molecules in form of a protein dimer. To test this hypothesis, three versions of the MiaA protein and seven species of tRNA substrates were purified. Binding studies by gel mobility shift assays, filter binding assays and gel filtration shift assays support the hypothesis that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ as a protein dimer but as a monomer to the anticodon stem-and-loop. These results were further supported by using steady state enzyme kinetic studies.^ The third hypothesis tested in this work was that the miaB gene in E. coli exists and is clonable. The miaB::Tn10dCm insertion mutation of Salmonella typhimurium was transduced to E. coli K-12 cells by using P$\sb1$ and P$\sb{22}$ bacteriophages. The insertion was confirmed by HPLC analyses of nucleotide profiles of miaB mutants of E. coli. The insertion mutation was cloned and DNA sequences adjacent to the transposon were sequenced. These DNA sequences were 86% identical to the f474 gene at 14.97 min chromosome of E. coli. The f474 gene was then cloned by PCR from the wild-type chromosome of E. coli. The recombinant plasmid complemented the mutant phenotype of the miaB mutant of E. coli. These results support the hypothesis that the miaB gene of E. coli exists and is clonable. In summary, functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli cells are further delineated in this work in perspectives of adaptation to stressful environmental conditions and protein:tRNA interaction. (Abstract shortened by UMI.) ^

Significance of tissue transglutaminase expression in multidrug resistant MCF -7 human breast cancer cells

Tissue transglutaminase (tTGase) is an enzyme that catalyzes the posttranslational modification of proteins via Ca2+-dependent cross-linking reactions. In this study, we extended our earlier observation that tTGase is highly expressed in MCF-7 human breast carcinoma cells selected for the multidrug resistance phenotype (MCF-7/DOX). To directly assess the involvement of tTGase in drug resistance, parental MCF-7 (MCF-7/WT) cells were transfected with cDNAs encoding either a catalytically active (wildtype) or inactive (mutant) tTGase protein. Expression of wildtype tTGase led to spontaneous apoptosis in MCF-7/WT cells, while the mutant tTGase was tolerated by the cells but did not confer resistance to doxorubicin. Analysis of calcium by a spectrofluorometric technique revealed that MCF-7/DOX cells exhibit a defective mechanism in intracellular calcium mobilization, which may play a role in preventing the in situ activation of tTGase and thus allowing the cells to grow despite expressing this enzyme. An elevation in intracellular calcium by treatment with the calcium ionophore A23187 induced rapid and substantial apoptosis in MCF-7/DOX cells as determined by morphological and biochemical criteria. Pretreatment of MCF-7/DOX cells with a tTGase-specific inhibitor (monodansylcadaverine) suppressed A12387-induced apoptosis, suggesting the possible involvement of tTGase-catalyzed protein cross-linking activity. A23187-induced apoptosis in MCF-7/DOX cells was further characterized by PARP cleavage and activation of downstream caspases (-3, -6, and -7). Another interesting aspect of tTGase/A23187-induced apoptosis in MCF-7/DOX cells was that these cells failed to show any prototypic changes associated with the mitochondrial (altered membrane potential, cytochrome c release, caspase-9 activation), receptor-induced (Bid cleavage), or endoplasmic reticulum-stressed (caspase-12 activation) apoptotic pathways. In summary, our data demonstrate that, despite being highly resistant to conventional chemotherapeutic drugs, MCF-7/DOX cells are highly sensitive to apoptosis induced by increased intracellular calcium. We conclude that tTGase does not play a direct role in doxorubicin resistance in MCF-7/DOX cells, but may play a role in enhancing the sensitivity of these cells to undergo apoptosis. ^

Proceedings of the Seventeenth Annual Biochemical Engineering Symposium

This is the seventeenth of a series of symposia devoted to talks by students about their biochemical engineering research. The first, third, fifth, ninth, twelfth, and sixteenth were at Kansas State University, the second and fourth were at the University of Nebraska-Lincoln, the sixth was in Kansas City and was hosted by Iowa State University, the seventh, tenth, thirteenth, and seventeenth were at Iowa State University, the eighth and fourteenth were at the University of Missouri–Columbia, and the eleventh and fifteenth were at Colorado State University. Next year's symposium will be at the University of Colorado. Symposium proceedings are edited by faculty of the host institution. Because final publication usually takes place elsewhere, papers here are brief, and often cover work in progress. ContentsThe Effect of Polymer Dosage Conditions on the Properties of ProteinPolyelectrolyte Precipitates, K. H. Clark and C. E. Glatz, Iowa State University An Immobilized Enzyme Reactor/Separator for the Hydrolysis of Casein by Subtilisin Carlsberg, A. J. Bream, R. A. Yoshisato, and G. R. Carmichael, University of Iowa Cell Density Measurements in Hollow Fiber Bioreactors, Thomas Blute, Colorado State University The Hydrodynamics in an Air-Lift Reactor, Peter Sohn, George Y. Preckshot, and Rakesh K. Bajpai, University of Missouri–Columbia Local Liquid Velocity Measurements in a Split Cylinder Airlift Column, G. Travis Jones, Kansas State University Fluidized Bed Solid Substrate Trichoderma reesei Fermentation, S. Adisasmito, H. N. Karim, and R. P. Tengerdy, Colorado State University The Effect of 2,4-D Concentration on the Growth of Streptanthus tortuosis Cells in Shake Flask and Air-Lift Permenter Culture, I. C. Kong, R. D. Sjolund, and R. A. Yoshisato, University of Iowa Protein Engineering of Aspergillus niger Glucoamylase, Michael R. Sierks, Iowa State University Structured Kinetic Modeling of Hybidoma Growth and Monoclonal Antibody Production in Suspension Cultures, Brian C. Batt and Dhinakar S. Kampala, University of Colorado Modelling and Control of a Zymomonas mobilis Fermentation, John F. Kramer, M. N. Karim, and J. Linden, Colorado State University Modeling of Brettanomyces clausenii Fermentation on Mixtures of Glucose and Cellobiose, Max T. Bynum and Dhinakar S. Kampala, University of Colorado, Karel Grohmann and Charles E. Yyman, Solar Energy Research Institute Master Equation Modeling and Monte Carlo Simulation of Predator-Prey Interactions, R. 0. Fox, Y. Y. Huang, and L. T. Fan, Kansas State University Kinetics and Equilibria of Condensation Reactions Between Two Different Monosaccharides Catalyzed by Aspergillus niger Glucoamylase, Sabine Pestlin, Iowa State University Biodegradation of Metalworking Fluids, S. M. Lee, Ayush Gupta, L. E. Erickson, and L. T. Fan, Kansas State University Redox Potential, Toxicity and Oscillations in Solvent Fermentations, Kim Joong, Rakesh Bajpai, and Eugene L. Iannotti, University of Missouri–Columbia Using Structured Kinetic Models for Analyzing Instability in Recombinant Bacterial Cultures, William E. Bentley and Dhinakar S. Kompala, University of Colorado

Noble metal abundances in ODP Leg 115 basalts (Table 1)

A comparison of 50 basalts recovered at Sites 706, 707, 713, and 715 along the Reunion hotspot trace during Ocean Drilling Program Leg 115 in the Indian Ocean shows that seafloor alteration had little effect on noble metal concentrations (Au, Pd, Pt, Rh, Ru, and Ir), determined by inductively coupled plasma-mass spectrometry (ICP-MS), which generally tend to decrease with magma evolution. Their compatible-element behavior may be related to the precipitation of Ir-Os-based alloys, chromite, sulfides, and/or olivine and clinopyroxene in some combination. The simplest explanation indicates silicate control of concentrations during differentiation. Basalts from the different sites show varying degrees of alkalinity. Noble metal abundances tend to increase with decreasing basalt alkalinity (i.e., with increasing percentages of mantle melting), indicating that the metals behave as compatible elements during mantle melting. The retention of low-melting-point Au, Pd, and Rh in mantle sulfides, which mostly dissolve before significant proportions of Ir-Os-based alloys melt, explains increasing Pd/Ir ratios with decreasing alkalinity (increasing melting percentages) in oceanic basalts. High noble metal concentrations in Indian Ocean basalts (weighted averages of Au, Pd, Rh, Pt, Ru, and Ir in Leg 115 basalts are 3.2, 8.1, 0.31, 7.3, 0.22, and 0.11 ppb, respectively), compared with basalts from some other ocean basins, may reflect fundamental primary variations in upper- mantle noble metal abundances

X-ray fluorescence (XRF) scannings and grain size analysis at sites in the Gulf of Cádiz

Contourites in the Gulf of Cádiz preserve a unique archive of Mediterranean Outflow Water (MOW) variability over the past 5.3 Ma. In our study we investigate the potential of geochemical data obtained by XRF scanning to decipher bottom current processes and paleoclimatic evolution at two different sites drilled through contourite deposits in the northern Gulf of Cadiz: Site U1387, which is bathed by the upper MOW core, and Site U1389, located more proximal to the Straits of Gibraltar. The lack of major downslope transport at both locations during the Pleistocene makes them ideal locations for the purpose of our study. The results indicate that the Zr/Al ratio, representing the relative enrichment of heavy minerals (zircon) over less dense alumosilicates under strong bottom current flow, is the most useful indicator for a semi-quantitative assessment of current strength. While most elements are biased by current-related processes, the bromine record, representing organic content, preserves the most pristine climate signal rather independent of grain size changes. Hence, Br can be used for chronostratigraphy and site-to-site correlation in addition to stable isotope stratigraphy. Based on these findings we reconstructed MOW variability for Marine Isotope Stages 1-5 using the Zr/Al ratio from Site U1387. The results reveal abrupt, millennial-scale variations of MOW strength during Greenland Stadials (GS) and Interstadials (GI) with strong MOW during GS and glacial Terminations and a complex behavior during Heinrich Stadials. Millennial-scale variability persisting during periods of poorly expressed GS/GI cyclicities implies a strong internal oscillation of the Mediterranean/North Atlantic climate system.

Analyses of lake La Thuile sediment core

Lake La Thuile, in the Northern French Prealps (874 m a.s.l.), provides an 18 m long sedimentary sequence spanning the entire Lateglacial/Holocene period. The high resolution multi-proxy (sedimentological, palynological, geochemical) analysis of the uppermost 6.2 meters reveals the Holocene dynamics of erosion in the catchment in response to landscape modifications. The mountain belt is at relevant altitude to study past human activities and the watershed is sufficiently disconnected from large valleys to capture a local sedimentary signal. From 12,000 to 10,000 cal. BP (10 to 8 ka cal. BC), the onset of hardwood species triggered a drop in erosion following the Lateglacial/Holocene transition. From 10,000 to 4500 cal. BP (8 to 2.5 ka cal. BC), the forest became denser and favored slope stabilization while erosion processes were very weak. A first erosive phase was initiated at ca . 4500 cal. BP without evidence of human presence in the catchment. Then, the forest declined at approximately 3000 cal. BP, suggesting the first human influence on the landscape. Two other erosive phases are related to anthropic activities: approximately 2500 cal. BP (550 cal. BC) during the Roman period and after 1600 cal. BP (350 cal. AD) with a substantial accentuation in the Middle Ages. In contrast, the lower erosion produced during the Little Ice Age, when climate deteriorations are generally considered to result in an increased erosion signal in this region, suggests that anthropic activities dominated the erosive processes and completely masked the natural effects of climate on erosion in the late Holocene.

Stable oxygen isotope record of Globigerinoides ruber of ODP Site 133-820 off the Great Barriere Reef

Oxygen-isotope ratio measurements are presented for the planktonic species Globigerinoides ruber collected from shallow-water, upper-slope sediments from Holes 820A and 820B in 280 m of water, on the seaward edge of the Great Barrier Reef. Correlation of the Site 820 isotope curve with deep-sea reference curves of the Pacific Ocean (Core V28-238, Hole 677A, Hole 607A) permits the definition of isotope stages 1 to 19 in the top 145 m of Holes 820A and 820B. However, paleontological data indicate that stages 4 and 7 might be missing and that two hiatuses occur at a depth of 8.05 to 12.1 and 34.55 to 35.8 mbsf. Using deep-sea Hole 677A as a reference for ice-volume variations, we determine the difference in isotopic signature between it and Site 820. We propose that this difference is a regional signal representing a progressive 4°C increase in surface-water temperature at Site 820. The proposed temperature change was initiated at about 400 k.y. and corresponds to a change from high-to-low frequency variations in Pleistocene isotope signals. We postulate that these changes may have catalyzed the growth of the Great Barrier Reef. The shift also coincides with changes in seismic character and some physical and chemical sediment characteristics.

Solid phase phosphorus geochemistry in surface sediments of sediment cores GeoB3718-4, GeoB3702-2 and GeoB3707-4

In this study we investigate benthic phosphorus cycling in recent continental margin sediments at three sites off the Namibian coastal upwelling area. Examination of the sediments reveals that organic and biogenic phosphorus are the major P-containing phases preserved. High Corg/Porg ratios just at the sediment surface suggest that the preferential regeneration of phosphorus relative to that of organic carbon has either already occurred on the suspension load or that the organic matter deposited at these sites is already rather refractory. Release of phosphate in the course of benthic microbial organic matter degradation cannot be identified as the dominating process within the observed internal benthic phosphorus cycle. Dissolved phosphate and iron in the pore water are closely coupled, showing high concentrations below the oxygenated surface layer of the sediments and low concentrations at the sediment-water interface. The abundant presence of Fe(III)-bound phosphorus in the sediments document the co-precipitation of both constituents as P-containing iron (oxyhydr)oxides. However, highly dissolved phosphate concentrations in pore waters cannot be explained, neither by simple mass balance calculations nor by the application of an established computer model. Under the assumption of steady state conditions, phosphate release rates are too high as to be balanced with a solid phase reservoir. This discrepancy points to an apparent lack of solid phase phosphorus at sediment depth were suboxic conditions prevail. We assume that the known, active, fast and episodic particle mixing by burrowing macrobenthic organisms could repeatedly provide the microbially catalyzed processes of iron reduction with authigenic iron (oxyhydro)oxides from the oxic surface sediments. Accordingly, a multiple internal cycling of phosphate and iron would result before both elements are buried below the iron reduction zone.

Dissolved chemical elements in river waters of the Wrangel Island

Pioneer information about chemical composition of river waters in the Wrangel Island has been obtained. It is shown that water composition reflects the lithogeochemical specifics of primary rocks and ore mineralization. In contrast to many areas of the Russian Far North river waters of the island are characterized by elevated background value of total mineralization (i.e., total dissolved solids, TDS) (0.3-2 g/l) and specific chemical type (SO4-Ca-Mg). This is related to abundance of Late Carboniferous gypsiferous and dolomitic sequences in the mountainous area of the island. It has also been established that salt composition of some streams is appreciably governed by supergene alterations of sulfide mineralization associated with quartz-carbonate vein systems. They make up natural centers of surface water contamination. Waters in such streams are characterized by low pH values (2.4-5.5), high TDS (up to 6-23 g/l) and SO4-Mg composition. These waters are also marked by anomalously high concentrations of heavy and non-ferrous metals, as well as REE, U, and Th.

Seasonally aggregated values of dissolved inorganic phosphorus in soil solution of the Jena Experiment (Main Experiment, year 2006)

This data set contains measurements of inorganic phosphorus in samples of soil solution collected in 2006 from the main experiment plots of a large grassland biodiversity experiment (the Jena Experiment; see further details below) that have been aggregated to seasonal values. In the main experiment, 82 grassland plots of 20 x 20 m were established from a pool of 60 species belonging to four functional groups (grasses, legumes, tall and small herbs). In May 2002, varying numbers of plant species from this species pool were sown into the plots to create a gradient of plant species richness (1, 2, 4, 8, 16 and 60 species) and functional richness (1, 2, 3, 4 functional groups). Plots were maintained by bi-annual weeding and mowing. Glass suction plates with a diameter of 12 cm, 1 cm thickness and a pore size of 1-1.6 µm (UMS GmbH, Munich, Germany) were installed in April 2002 in depths of 10, 20, 30 and 60 cm to collect soil solution. Manual soil matric potential measurements were used to regulate the vacuum system. Manual soil matric potential measurements were used to regulate the vacuum system. The sampling bottles were continuously evacuated to a negative pressure between 50 and 350 mbar, such that the suction pressure was about 50 mbar above the actual soil water tension. Thus, only the soil leachate was collected. Cumulative soil solution was sampled biweekly and analyzed for dissolved inorganic P (PO4P). Here volume-weighted mean values are provided as aggregated seasonal values (spring = March to May, summer = June to August, fall = September to November, winter = December to February) for 2006 in spring. To calculate these values, the sampled volume of soil solution is used as weight for P concentrations of the respective sampling date. Inorganic phosphorus concentrations in the soil solution were measured photometrically with a continuous flow analyzer (CFA Autoanalyzer [Bran&Luebbe, Norderstedt, Germany]). Ammonium molybdate catalyzed by antimony tartrate reacts in an acidic medium with phosphate and forms a phospho-molybdic acid complex. Ascorbic acid reduces this complex to an intensely blue-colored complex. As the molybdic complex forms under strongly acidic conditions, we could not exclude the hydrolysis of labile organic P compounds in our samples. Furthermore, the molybdate reaction is not sensitive for condensed phosphates. The detection limits of both TDP and PO4P were 0.04 mg P l-1 (Autoanalyzer, Bran&Luebbe).