4. Předn. 3.12. 2013 Chemie, struktura a interakce nukleových kyselin Fyzikální vlastnosti a izolace DNA Denaturace, renaturace a hybridizace DNA Biosyntetické polynukleotidy V posledních letech jsou k dispozici komerčně dostupné kolonky využívající imobilizaci DNA na pevném podkladu. K separaci DNA jsou rovněž používány magnetické kuličky (magnetic beads) Characterize your DNA sample: ds x ss, circular x linear circular: nicked, oc; covalently closed, cc, cd linear: cohesive or blunt ends number of base pairs, ssb purity: protein, RNA .... content analytical methods Unusual bases, DNA methylation Denaturation x degradation aggregation renaturation/hybridization DNA DENATURATION and RENATURATION/HYBRIDIZATION J. Marmur and P. Doty DNA renaturation/reassociation depends on the concentration of the DNA molecules and the time allowed for reassociation. Often imperfect matches may be formed which must again dissociate to allow the strands to align correctly. C0t value of DNA is defined as the initial concentration C0 in moles nucleotides per Litre multiplied by time t in seconds. C0t reflects complexity of DNA. Methods: S1, hydroxyapatite - dsDNA binds more strongly Důležité modely vlivu sekvence nukleotidů na vlastností DNA nukleosid-difosfáty nevyžaduje primer ani matrici nukleosid-trifosfáty poly(A) poly(rC) poly(dG) poly(U) poly(rT) Mirror Molecules NA bases x L-amino acids (aa) x D-carbohydrates D-aa already known in the late 1800‘s In chemistry of life L-aa were the rule (only gly achiral) Ribosomes compatible only with L-aa but not with D-aa For a long time the only exceptions to this pattern were found in bacteria. Recently biologically active D-aa’s have been found to perform important roles in human physiology. 1990’s S. Snyder D-compounds serve as neurotransmitters „Like most of science, whenever there is something really new or different, some people say some people say: That’s ridiculous“ Later: D-aspartate shown to be a neurotransmitter involved in normal brain development D-serine teams up with L-glutamate to activate neuronal molecules essential for synaptic plasticity – key to learning and forming memories 2002 P.Kuchel: Platypus poison contains D-aa 2009 D-aa‘s play unexpected function in bacterial cell walls 2010 complex assemblies of bacteria use D-aa when the biofilms should disperse D-serine important factor in schizofrenia (lower amount of this aa) x higher amounts: increase brain damage in stroke Review 2012 Since d-amino acids were identified in mammals, d-serine has been one of the most extensively studied "unnatural amino acids". This brain-enriched transmitter-like molecule plays a pivotal role in the human central nervous system by modulating the activity of NMDA receptors. Physiological levels of d-serine are required for normal brain development and function; thus, any alterations in neuromodulator concentrations might result in NMDA receptor dysfunction, which is known to be involved in several pathological conditions, including neurodegeneration(s), epilepsy, schizophrenia, and bipolar disorder. In the brain, the concentration of d-serine stored in cells is defined by the activity of two enzymes: serine racemase (responsible for both the synthesis and degradation) and d-amino acid oxidase (which catalyzes d-serine degradation). Both enzymes emerged recently as new potential therapeutic targets for NMDA receptor-related diseases. In this review we have focused on human d-amino acid oxidase and provide an extensive overview of the biochemical and structural properties of this flavoprotein and their functional significance. Furthermore, we discuss the mechanisms involved in modulating enzyme activity and stability with the aim to substantiate the pivotal role of d-amino acid oxidase in brain d-serine metabolism in physiological and pathological conditions and to highlight its great significance for novel drug design/development. Only L-aa are produced in cells Brain cells produce an enzyme that flips the handedness of L-serine to its D-form Platypus venom is made in a similar way: ribosome builds up the peptide from regular L-aa. Then an enzyme flips an aa into its D-form 2005 Kreil (Austrian Academy of Sciences, Wien) making D-aa’s in tree frog venom. Without a single D-aa, the peptide has no hallucinogenic effect D-aa’s found in poisons of a wide range of organisms but these aa’s have also more peaceful purposes, e.g. in lobsters they keep salt levels in order Biggest users of D-aa’s are microbes For example, peptidoglycan in cell walls may contain D-ala, D-met or D-leu Important research task: Understanding how bacteria exploit D-aa’s for communication Attempts to develop drugs that break-up the biofilms in our teeth, in the lungs of cystic fibrosis or in medical equipment Addition of D-aa’s to therapeutic peptides or proteins may prevent their enzymatic degradation Speculations: D-aa’s in bacterial cells living on our skin or elsewhere in the body might be important for our well-being and behavior Preliminary reports: Yoko Nagata, Tokyo: D-aa in human saliva D-ala in the insulin-secreting beta cells in rats Enzymes converting L-aa into D-aa found in human and rat hearts. Precise role of such enzyme in human physiology is still “a total mystery”. SOME LITERATURE The New Ambidextrous Universe: Symmetry and Asymmetry from Mirror Reflectionst to Superstrings. Third revised edition. Martin Gardner. Dover, 2005. High Dose D-Serine in the Treatment of Schizofrenia. Josua Kantrowitz et al. in Schizophrenia Research, Vol. 121, No. 1, pages 125-130; August 2010. www.ncbi.nlm.nih.gov/pmc/articles/PMC3111070 D-Amino Acids in Chemistry, Life Sciences, and Biotechnology. Edited by Hans Brückner and Noriko Fujii. Wiley, 2011. Emerging Knowledge of Regulatory Roles of D-Amino Acids in Bacteria. Felipe Cava et al. in Cellular and Molecular Life Sciences, Vol. 68, No. 5, pages 817-831; March 2011. www.ncbi.nlm.nih.gov/pmc/articles/PMC3037491 The role of D-amino acids in amyotrophic lateral sclerosis pathogenesis: a review Author(s): Paul, Praveen; de Belleroche, Jacqueline Source: AMINO ACIDS Volume: 43 Issue: 5 Pages: 1823-1831 DOI: 10.1007/s00726-012-1385-9 Published: NOV 2012 Structure-function relationships in human d-amino acid oxidase Author(s): Sacchi, Silvia; Caldinelli, Laura; Cappelletti, Pamela; et al. Source: AMINO ACIDS Volume: 43 Issue: 5 Pages: 1833-1850 DOI: 10.1007/s00726-012-1345-4 Published: NOV 2012 Times Cited: 2 (from Web of Nutritional and medicinal aspects of D-amino acids Author(s): Friedman, Mendel; Levin, Carol E. Source: AMINO ACIDS Volume: 42 Issue: 5 Pages: 1553-1582 DOI: 10.1007/s00726-011-0915-1 Published: MAY 2012 Lokální struktury DNA a metody jejich analýzy Parametry různých typů ds DNA Metody analýzy lokálních struktur DNA Ohyby v DNA Typy lokálních struktur stabilizovaných nadšroubovicovým vinutím Strukturní rozhraní Výskyt lokálních struktur DNA in vivo Polymorphy of the DNA double helix B. sublilis and B. brevis DNAs have the same G+C content and different nucleotide sequence B. subtilis B. brevis DNA structures from X-ray crystal analysis DNA double helix is polymorphic depending on the nucleotide sequence MICROHETEROGENEITY OF THE DNA DOUBLE HELIX FORMS Studies of the detailed relationships between nucleotide sequence and DNA structure became feasible by the end of the 70s, when organic synthesis had been developed to the point where oligodeoxynucleotides (ODN) could be produced in the purity and quantity necessary for the preparation of single crystals for X-ray diffraction (and NMR) studies. Three main families of DNA forms were identified by crystallographic analysis of ODN: right-handed A and B-forms and the left-handed Z-form. B-, A- and Z-helices The A-, B- and Z-helices have distinctly different shapes which are due to the specific positioning and orientation of the bases with respect to the helix axis. In A-DNA, the base pairs are displaced from the helix axis, the major groove is very deep, and the minor groove is very shallow. In B-DNA the major and minor grooves are of similar depths and the helix axis is close to the base pair center. In Z-DNA the minor groove is deep and the major groove is convex. In A- and B-DNA a single nucleotide can be considered as the repeat unit, while in Z-DNA the repeat unit is a dinucleotide. In A-duplexes base pairs are heavily tilted in contrast to base pairs in B-duplexes which are almost perpendicular to the helical axis. (Table 1). Many of the structural differences between the helices arise from the puckering of the sugar ring; C3'-endo is typical for A-DNA, while in Z-DNA C3'-endo alternates with C2'-endo. In B-DNA sugar pucker tends to favor the C2'-endo or C1'-exo,but the distribution of conformations is much broader than in A- and Z-DNA. A B Z The right-handed A- and B-forms have the anti glycosidic bond, whereas in the left-handed Z-helix the orientation alternates between syn (for purines) and anti (for pyrimidines). In the latter structure the orientation around the C4'-C5' bond with respect to the C3' atom alternates between gauche+ and trans conformations for cytidine and guanosine, respectively. The alternating features of Z-DNA result in the zig-zag shape of its sugar-phosphate backbone, from which the name was derived. The changes in the backbone and glycosidic-bond conformations are accompanied by substantial variations in the stacking interactions between successive base pairs in Z-DNA. Methylation or bromination of cytosines at position 5 (studied mainly in ODNs with alternating C-G sequence) stabilizes Z-DNA. Under certain conditions even non-alternating sequences of purines and pyrimidines can assume the conformation of Z-DNA with thymines in a syn orientation. The outer surface features of such a Z-helix are different at the non-alternating sites but the backbone is similar to that observed with alternating sequences. Single-strand selective chemical probes of the DNA structure DNA structures from X-ray crystal analysis DNA double helix is polymorphic depending on the nucleotide sequence DNA footprinting mapping of DNA interactions Enzymatic probe (DNAase I) Chemical probe - DEPC Lokální struktury DNA a metody jejich analýzy Parametry různých typů ds DNA Metody analýzy lokálních struktur DNA Ohyby v DNA Typy lokálních struktur stabilizovaných nadšroubovicovým vinutím Strukturní rozhraní Výskyt lokálních struktur DNA in vivo Metody analýzy lokálních stuktur DNA Single-strand selective chemical probes of the DNA structure Discovery of the cruciform in sc DNA D M J LILLEY, 1981 44 LEFT-HANDED Z-DNA alternating pu-py CRUCIFORM inverted repeat CURVATURE 4-6 A’s in phase with the helix turns SINGLE-STRANDED region AT-rich Text TRIPLEX structure homopu.homopy HAIRPIN SUPERCOIL Negative SUPERCOILING stabilizes local DNA structures Physical methods such as NMR and X-ray analysis indispensable in the research of linear DNA structures are of limited use in studies of local structures stabilized by supercoiling INVERTED REPEAT DNA B-Z junction Strukturní rozhraní mezi B- a Z-DNA detekce pomocí chemické strukturni sondy konec/12.11.08 17.18. J. Šponer - mat. modelování NA (ca 45 min) DNA triplexes their identification by chemical probes INTERmolecular INTRAmolecular pu py 1990 existence of triplex H-DNA in cells was demostrated in Paleček’s laboratory Homopy.homopy sekvence se zrcadlovou symetrii je potřebná pro vznik triplexu H-DNA *H-DNA může vzniknout i v jiných sekvencích E. Trifonov, Weizmann Inst, Rehovot M. Frank-Kamenetskii, Boston Univ S. Mirkin, Univ. Illinois, Chicago D. Lyamichev ........... všichni původně v Moskvě G-quartet DOPLNĚK k předn. prof. M. Vorlíčkové DIRECT vs. INVERTED REPEATS Výskyt lokálních struktur DNA v prokaryotních a eukaryotních buňkách Proc. Natl. Acad. Sci. USA 87 (1990) 8373-8377 Chemická modifikace DNA v buňkách pomocí komplexu OsO4 (Os,bipy) a její využití pro testování -σ DNA Strukturní přechod DNA duplex – křížová forma v buňce může informovat o superhelikální hustotě DNA a o jejich změnách působených změnami prostředí nebo genetickými faktory Salt shock Cruciform structures in E. coli cells TRIPLEX DNA V BUŇKÁCH PROKÁZANÝ POMOCÍ Os,bipy P. Karlovský, P. Pecinka, M. Vojtísková, E. Makaturová, E. Palecek, FEBS Letters 274 (1990)39-42 Konformer H-y5 pravděpodobně v buňkách převažuje Unconstrained supercoiling in eukaryotic cells In difference to the prokaryotic genome the eukaryotic genome was for years believed not to be under the superhelical stress due to the accommodation of the DNA writhing around histone octamers in nucleosomes (Pearson, 1996, Van Holde, 1994). The actively transcribing portion of the eukaryotic genome was, however, shown to contain unconstrained supercoiling, part of which can be attributed to the process of transcription per se. Using prokaryotic cells it has been recently shown that the effects of transcriptionally driven supercoiling are remarkably large scale in vivo(in a kbp range). Similarly to the transcription effects, in DNA replication intermediates supercoils are formed both behind and in front of the replication fork and superhelical stress is distributed throughout the entire partially replicated DNA molecule. and gyrase activity of topoisomerases. Unconstraint negative supercoiling stabilizes local DNA structures such as cruciforms, Z-DNA segments and intramolecular triplexes. Mounting evidence of the existence of these structures in vivo both in prokaryotic and eukaryotic cells has been reviewed. It appears that alternative DNA structures are located in extranucleosomal regions such as linkers and DNase hypersensitive site but probably not within the DNA wrapped around DNA octamer. EP et al, Oncogene 2004 Disociace nukleosomu vede k aktivaci transkripce a tvorbě negativní nadšroubovice (supercoil) schopné indukovat lokální struktury. Transkripce může být aktivována vazbou specif. proteinu (např. Z-binding ) posunující rovnováhu ve prospěch lokální struktury, následované vznikem pozitivní nadšroubovice a ztrátou nukleosomu 12.11. - 6. předn. a. DR. L. HAVRAN: SYNTHESA OLIGONUKLEOTIDŮ (ca. 10 min) b. Prof. M. VORLIČKOVÁ: CD DNA (a 40 min)