Chemie, struktura a interakce nukleových kyselin 2009 5.-6.PŘEDNÁŠKA EP 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 Zhelix are different at the non-alternating sites but the backbone is similar to that observed with alternating sequences. Local DNA Structures and Nucleotide Sequence The significant variations in some of the global parameters (Table 2), dependent on nucleotide sequence, result in local changes along the DNA double helix. Such relations have been analyzed in detail by several authors and reviewed by Shakked and Rabinovich. In A- and B-DNA these variations seem to be determined mainly by the specific interactions between the stacked base pairs and also to some extent by neighboring bases. In particular, homopolymer dinucleotide steps show a wide spectrum of stacking characteristics which are markedly neighbor dependent. On the other hand pyrimidine-purine steps in A-DNA (especially the C-G steps) often display a low twist and high slide that are only slightly dependent on neighboring steps. In ZDNA the shape of the helix surface changes significantly due to deviations in the regular alternation of the purine-pyrimidine sequence while the sugar-phosphate backbone does not change. The effect of the nucleotide sequence on the fine geometrical features of each DNA form has been clearly demonstrated but not fully elucidated. The emerging rules should, however, be considered as tentative since they were based on a relatively small number of examples. The well-known "Calladine's rules" are now perceived to be incomplete and to neglect important factors other than the steric clash of purine rings. DNA Hydration Information about the organization of water molecules in DNA forms has been gained from X-ray diffraction analysis of crystals. Distinct hydration patterns were observed in the major and minor grooves and around the sugar-phosphate backbone. It was proposed that in DNA with a mixed nucleotide sequence hydration of the backbone is related to global conformation. In A- and Z-DNA a chain of water molecules can bridge the phosphate oxygens along the backbone. There are more water molecules around each phosphate group in the B-DNA but almost no water bridges between the phosphate oxygens, as the distances between phosphate oxygens in this DNA form are too great to be linked with a single water molecule. It appears that specific nucleotide sequences that create local changes in the DNA double helix may also affect the backbone hydration pattern. Even greater dependence of the hydration patterns on nucleotide sequence has been found in the DNA grooves. In A-DNA specific hydration patterns occur in the major grooves. A string of well ordered water molecules hydrogen bonded to oxygen and nitrogen atoms in the minor groove has been found in the central AATT sequence of the B-DNA dodecamer d(CGCGAATTCGCG). This specific hydration of B-DNA, called "spine of hydration", significantly contributes to DNA stability. Studies of further B-DNA helices revealed two ribbons of water molecules along the walls of wide regions of the minor groove while narrow regions of the minor groove contained an ordered zig-zag spine of hydration. It appears that the interdependence between nucleic acid structure and the solvent represents one of the bases for DNA double helix polymorphy. Metody analýzy lokálních stuktur DNA DNA footprinting mapping of DNA interactions Enzymatic probe (DNAase I) Chemical probe - DEPC Single-strand selective chemical probes of the DNA structure Discovery of the cruciform in sc DNA D M J LILLEY, 1981 12.11. - 6. předn. a. DR. L. HAVRAN: SYNTHESA OLIGONUKLEOTIDŮ (ca. 10 min) b. Prof. M. VORLIČKOVÁ: CD DNA (a 40 min) 6. Přednáška-9.12.09 24 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 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 ........... S. Lazurkin všichni původně v Moskvě G-quartet DOPLNĚK k předn. prof. M.Vorlíčkové 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 - 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. 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 Recently mechanisms by which the effect of supercoil-stabilized non-B DNA structures on transcription can be exerted in eukaryotic cells have been proposed. If the promoter region of the gene is blocked by a nucleosome and sites for transcription factors are inaccessible, nucleosome dissociation can result in transcriptional activation provided transcription factors are available; such dissociation is accompanied by formation of negative supercoils capable to induce alternative structure in a linker. Similar transcriptional activation may be produced by binding of a specific protein, shifting the equilibrium in favor of an alternative DNA structure, followed by formation of positive supercoils and loss of a nucleosome. For this mechanism, denominated as conformational compensation, precise location or orientation of potential non-B sequence is not critical. Another mechanism based on binding of proteins to potential non-B sequences has been proposed by Hatfield et al. Supercoiling can locally destabilize B-DNA structure and drive transitions to other structures at susceptible sequences. In principle the supercoil-driven local structural transitions can be either inhibited or facilitated by proteins that bind at or near potential transition sites. If a DNA segment, susceptible to forming a supercoil-induced alternative structure, is stabilized in the B-form by a DNAbinding protein, the propensity of this segment for structural transition will be within the same DNA domain. Positioning of this site in the promoter region may facilitate open complex formation and activation of gene expression.