ISBN: 978-83-60043-10-3 - eurobic9

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Eurobic9, 2-6 September, 2008, Wrocław, Poland P151. Artificial Ribonucleic Acids as Scaffolds for Mercury(II) Ions S. Paulus a , S. Johannsen a , N. Düpre b , J. Müller b , R.K.O. Sigel a a Institute of Inorganic Chemistry, University of Zurich, Winterthurstr. 190, 8057, Zurich, Switzerland, b Faculty of Chemistry, Dortmund University of Technology, Otto-Hahn-Str. 6, 44227, Dortmund, Germany e-mail: supaulus@aci.uzh.ch Metal-modified nucleic acids have become increasingly important as they provide scaffolds that can potentially be used for molecular wires. A DNA duplex with mismatched T-T pairs can bind Hg 2+ by forming T-Hg 2+ -T base pairs, which are as stable as a Watson-Crick base pair.[1] We are interested in the formation of an analogous array of Hg 2+ by using RNA instead.[2] The required single-stranded RNA sequences were synthesized by in vitro transcription, which yields the RNA in amounts sufficient for a detailed characterization by NMR. Indeed, T7 RNA polymerase successfully incorporates two to twenty consecutive uracil residues into RNA. In the presence of Hg 2+ , a structural rearrangement from hairpin to regular duplex by forming Hg 2+ - mediated base pairs has been characterized in detail for one of the sequences (see Figure). This rearrangement was verified by several techniques, e.g. NMR, DLS, UV and CD spectroscopy.[2] Ongoing investigations now focus on the incorporation of stretches of the chemically more stable thymine into RNA. For this purpose we are using the T7 RNA polymerase mutant Y639F, which does not discriminate deoxyribose against ribose sugars.[3] Indeed, we were able to construct RNAs containing poly T-sequences, which are presently subject to structural studies. Acknowledgement: Financial support by the European ERAnet-Chemistry, the Swiss National Science Foundation (20EC21-112708 and SNF-Förderungsprofessur PP002-114759 to R.K.O.S.) and the DFG (JM1750/2-1 and Emmy Noether programme JM1750/1-3 to J.M.) is gratefully acknowledged. References: [1] Y. Tanaka, S. Oda, H. Yamaguchi, Y. Kondo, C. Kojima, A. Ono, J. Am. Chem. Soc. 2007, 129, 244-245. [2] S. Johannsen, S. Paulus, N. Düpre, J. Müller, R.K.O. Sigel, J. Inorg. Biochem. 2008, <strong>10</strong>2, 1141-1151. [3] R. Sousa, R. Padilla, EMBO J. 1995, 14, 4609-4621. _____________________________________________________________________ 270
Eurobic9, 2-6 September, 2008, Wrocław, Poland P152. Quantitative Electrospray Mass Spectrometry of Zinc Finger Oxidation K. Piątek a , J. Smirnova b , L. Zhukova a , A. Witkiewicz-Kucharczyk a , E. Kopera a , J. Olędzki a , A. Wysłouch-Cieszyńska a , T. Schwerdtle c , A. Hartwig c , P. Paluma b , W. Bal a a Department of Biophysics, Institute of Biochemistry and Biophysics, PAS, Pawińskiego 5a, 02-<strong>10</strong>6, Warsawa, Poland, b Department of Gene Technology, Tallinn Technical University, Akadeemia tee 15, 12618 Tallinn, Tallinn, Estonia c Institute of Food Technology and Food Chemistry, Technical University Berlin, Gustav-Meyer-Allee 25, D-13355, Berlin, Germany e-mail: kpiatek@ibb.waw.pl Oxidation is a crucial inhibitor of zinc fingers (ZF) [1]. Using the Zn(II) complex of 37-peptide acetyl- DYVICEECGKEFMDSYLMNHFDLPTCDNCRDADDKHK-amide (ZnXPAzf), the Cys4 ZF of human DNA repair protein XPA, we developed an ESI MS approach for quantitative study of ZF oxidation kinetics. For this purpose we studied oxidation of ZnXPAzf by H2O2 using HPLC of covalent reaction products, PAR-based zinc release assay, and ESI MS. All yielded independently the same reaction rate, thus demonstrating quantitative applicability of ESI MS [2]. We then used this approach to study aerobic reactions of ZnXPAzf with Snitrosoglutathione (GSNO), arsenite and monomethylarsonous acid (MMA). GSNO initially formed a ZnXPAzf- GSNO complex, followed by thiol transnitrosylations and finally disulfide formation. These results showed that at low exposures GSNO may reversibly regulate the ZF, while transnitrosylation by GSNO, occurring at prolonged exposures, is damaging. For XPA this may lead to DNA repair inhibition [3]. MMA, but not arsenite, released Zn(II) from ZnXPAzf easily, forming arsenical thiol esters of XPAzf. Unprotected thiol groups were oxidized to disulfides. The estimated affinity of MMA to XPAzf is 30-fold higher than the values typical for arsenite binding to thiols, indicating a particular susceptibility of ZFs to MMA, and providing a novel molecular pathway in arsenic carcinogenesis [4]. In summary, ESI MS is a valuable tool for quantitative bioinorganic studies. References: [1] A Witkiewicz-Kucharczyk, W Bal, Damage of zinc fingers in DNA repair proteins, a novel molecular mechanism in carcinogenesis. Toxicol Lett 162, 29-42, 2006. [2] J Smirnova, L Zhukova, A Witkiewicz-Kucharczyk, E Kopera, J Olędzki, A Wysłouch-Cieszyńska, P Palumaa, A Hartwig, W Bal, Quantitative electrospray mass spectrometry of zinc finger oxidation: the reaction of XPA zinc finger with H2O2, Anal Biochem 369, 226-231, 2007. [3] J Smirnova, L Zhukova, A Witkiewicz-Kucharczyk, E Kopera, J Olędzki, A Wysłouch-Cieszyńska, P Palumaa, A Hartwig, W Bal, Reaction of the XPA zinc finger with GSNO, Chem Res Toxicol 21, 386-392, 2008. [4] K Piątek, T Schwerdtle, A Hartwig, W Bal, Monomethylarsonous acid destroys a tetrathiolate zinc finger much more efficiently than inorganic arsenite. Mechanistic considerations and consequences for DNA repair inhibition. Chem Res Toxicol 21, 600-606, 2008. _____________________________________________________________________ 271
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ISBN: 978-83-60043-10-3 - eurobic9

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