A468
Biochemical Society Transactions (2000) Volume 28, Part 5
1903 Stopped-flow fluorescence studies of DNA base flipping by HhaI methyltransferase S. Serva', E. Weinhold* and S . KlimaSauskas' 'Institute of Biotechnology. Vilnius, Lithuania 'MPlfnr Molekulare Physiologie. Donmumi, Germany
Rotation of a nucleotide outof the DNA helix (base flipping) has been first observed for theHhaI methyltransferase followed by numerous other DNA modification and repair enzymes. M.HhaI catalyzes transfer of a methyl group from cofactosadenosyl-L-methionineonto the C5 position of the first cytosine in the target sequence GZGC. In this study, stopped-flow and rapid-quench techniques were employed in combination with fluorescence detection for kinetic characterization of individual steps on the reaction pathway of M.HhaI. Selective labeling of the DNA substrate was achieved by synthetic incorporation of 2aminopurine at the target position which showed a dramatic increase in fluorescence signal upon transition of the base from the stacked to an extrahelical postition. Association, dissociation and single-turnover experimental setups permitted the direct determination of microscopic rate constants for DNA binding, flipping of the target base, methyl group transfer and release of methylated DNA. We demonstrate here that the target sites on short DNA duplexes are predominantly located via a diffusioncollision pathway and the subsequent base flipping is nearly instantaneous (< lms) for the G-ZAP mismatch. We show for the first time that the chemical methyl transfer step(0.15 s:') is faster than the steady-state turnover (0.02 s - ) and directly confirm that decay of the ternary product complex (methylated DNA-M.HhaI-AdoHcy) is rate-limiting in the catalytic cycle. Global fitting and simulation analysis in conjunction with structural data suggest detailed catalytic mechanisms for DNA base flipping and catalysis by DNA cytosine-5 methyItransferases.
1905 MOLECULAR HYBFUDIZATION OF DECATHYMIDYLATE MODIFIED WITH A PHENAZINE DERIVATIVE
Antonina Shcherbakova', Victor Zozulya', Yuri Blagoi', Igor Dubeq, Olesya Fedoryd, Dmitrij F e d o r y d 'Institute for Low Temperature Physics and Engineering, Nat. Acad. Sci. of Ukraine, 310164 Kharkov. Ukraine, 'Institute of Bioorganic Chemistry and Petrochemistry. Nat. Acad Sci. of Ukraine, 253660 Kiev, Ukraine
A covalent attachment of intercalating dyes to antisense and antigene oligonucleotides are used for stabilization of the double and triple helix formations. Usually the dye attachment is realized via a polymethylene chain. In this communication we present results of the investigation of another variant of dyeoligonucleotide conjugation. A nucleoside derivative of imidazophenazine, with a neutral chromophore, (Pzn) was covalently l i e d to the 3'-end of decathymidylate via a ribose residue of the dye ((dT)&n). An oligomer (dA)ls, polymers poly(rA) and poly(dA)poly(dT) were used as the targets for the hybridization. To reveal the stabilizing effects of P n , the results were compared with those obtained with unmodified (dT)m. The studies were carried out by the method of thermal denaturation using absorbance and fluorescence detections of melting transitions. The attached Pm strongly stabilized duplexes and triplexes formed by ( d T ) & n due to the intercalation of the dye chromophore into the dA, rA and dA.dT strands. Temperature of half dissociation was increased by 10-11 "C for the duplex complexes and by 15-20 OC for the triplex ones.
1906 Splice variants of human phosphatidylethanolamine 1904 Cadmium induced suppression in the activities of key metabolic enzymes and synthesis of stress specific proteins in growing rice (Oryzasnriva L.) seedlings Kavita Shah and R. S. Dubey, Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi 22 1005, India, Email:
[email protected] in Cadmium is a potentially toxic heavy metal pollutant present in soil in the vicinity of industrial areas. Our experiments suggest that when IOOpM ( ~ 1 1 . 2ppm) and 5 O O p M (=56 ppm) Cd is supplied as Cd(NO& to growing rice plants, 245-31 1 ppm Cd gets localized in roots and 56 to 134 ppm in shoots. Cd treated seedlings show decreased seedling vigour, chlorosis as well as excessive yellowing and broadening of leaves. Elevated levels of extractable proteins, total free amino acids more specially proline and RNA and inhibition in the activity of enzymes protease, RNase and organ specific changes in the behaviour of amino and carboxypeptidases are observed in Cd treated seedlings Inhibition in the activities of key phosphorolytic enzymes with a concomitant decline in the intensities of acid phosphatase isoforms and a lowering of P, level in different parts of Cd treated seedlings suggest a limitation in the availability of phosphate for Cd-grown plants. A 18kDa Cd-inducible protein complex containing 4-SH groups per protein molecule has been isolated which possibly helps in sequestration of excess Cd ions inside the rice plants These findings appear to contribute towards the toxicity of Cd for plants growing in metal polluted soils and are applicable to the study of bioremediation of metals from the soil environment
2000 Biochemical Society
N-methyltransferase (PEMT) and the regulation of phosphatidylcholine (FC) biosynthesis. D.J.Shields, L.B. Agellon, D.E. Vance. Dept. of Biochemistry. University of Alberta,Edmonton, AJ3 T6G 2S2. Canada. PC is the principal component of cellular membranes and is required for signal transduction, lipoproteins and bile acids. FC is produced in all nucleated cells by the CDP-choline pathway. An additional pathway exists in the liver where PEMT catalyzes three methylation reactions converting phosphatidylethanolamine to PC. Mice with a disrupted PEMT gene display increased activity of the CDP-choline pathway. Conversely, animals fed a choline-deficient diet that eliminates the initial substrate for the CDP-choline pathway have elevated PEMT transcription and activity. Hence, coordinate regulation of PC biosynthesis is modulated, at least in part, at the transcriptional level. Using a liver cDNA library we cloned three PEMT cDNAs. each of which has a unique fust untranslated exon. Cloning the full-length PEMT gene from a BAC library confirmed the integrity of the cDNAs. The human gene is at least 35kb in length and contains 9 exons. To characterize transcriptional regulation of human PEMT, we are constructing a transgenic mouse using the human gene. This animal will be crossed with our PEMT-I- mice thereby generating an animal model that only expresses human PEMT. Elucidation of the transcriptional regulation of PEMT will provide valuable insight to mechanisms regulating synthesis of the essential biomolecule. phosphatidylcholine.
