UV absorption and CD spectroscopy, along with polyacrylamide gel electrophoresis, were used to study conformational properties of DNA fragments containing the trinucleotide repeat (GCC)(n) (n = 4, 8 or 16), whose expansion is correlated with the fragile X chromosome syndrome. We have found that the conformational spectrum of the (GCC)(n) strand is wider than has been shown so far. (GCC)(n) strands adopt the hairpin described in the literature under a wide range of salt concentrations, but only at alkaline (>7.5) pH values. However, at neutral and slightly acid pH (GCC)(4) and (GCC)(8) strands homodimerize. Our data suggest that the homodimer is a bimolecular tetraplex formed by two parallel-oriented hairpins held together by hemi-protonated intermolecular C.C(+) pairs. The (GCC)(16) strand forms the same tetraplex intramolecularly. We further show that below pH 5 (GCC)(n) strands generate intercalated cytosine tetraplexes, whose molecularity depends on DNA strand length. They are tetramolecular with (GCC)(4), bimolecular with (GCC)(8) and monomolecular with (GCC)(16). i-Tetraplex formation is a complex and slow process. The neutral tetraplex, on the other hand, arises with fast kinetics under physiological conditions. Thus it is a conformational alternative of the (GCC)(n) strand duplex with a complementary (GGC)(n) strand.
- MeSH
- cirkulární dichroismus MeSH
- dimerizace MeSH
- DNA chemie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- konformace nukleové kyseliny * MeSH
- lidé MeSH
- spektrofotometrie ultrafialová MeSH
- syndrom fragilního X genetika MeSH
- trinukleotidové repetice * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
Conformational properties of microsatellite DNA regions are the probable reason of their expansions in genomes which lead to serious genetic diseases in some cases. Using CD spectroscopy, UV absorption spectroscopy and polyacrylamide gel electrophoresis, we study in this paper conformational properties of (CGA)(4) and compare them with those of (CAG)(4) - a related repeat, connected with Huntington's disease. We show that (CGA)(4) can adopt several distinct conformations in solution. Around neutral pH it forms a parallel-stranded homoduplex containing C(+).C, G.G, and A.A base pairs. Under the same conditions (CAG)(4) forms a hairpin. At slightly alkaline pH values and low ionic strength, (CGA)(4) also folded into a hairpin which transformed into a bimolecular anti-parallel homoduplex at increasing salt concentrations. The duplex easily isomerized into left-handed Z-DNA, implying that the mismatched adenines between G.C pairs facilitate rather than hinder the B-Z transition. No similar changes took place with (CAG)(4). Thus, the conformational repertoire of (CGA)(4) includes parallel, anti-parallel, right-handed, and left-handed homoduplexes. In contrast, (CAG)(4) invariably adopts only a single conformation, namely the very stable hairpin.
We took 28 fragments of DNA whose crystal structures were known and used CD spectroscopy to search for conditions stabilising the crystal structures in solution. All 28 fragments switched into their crystal structures in 60-80% aqueous trifluorethanol (TFE) to indicate that the crystals affected the conformation of DNA like the concentrated TFE. The fragments crystallising in the B-form also underwent cooperative TFE-induced changes that took place within the wide family of B-form structures, suggesting that the aqueous and crystal B-forms differed as well. Spermine and magnesium or calcium cations, which were contained in the crystallisation buffers, promoted or suppressed the TFE-induced changes of several fragments to indicate that the crystallisation agents can decide which of the possible structures is adopted by the DNA fragment in the crystal.
- MeSH
- cirkulární dichroismus * MeSH
- DNA chemie metabolismus MeSH
- konformace nukleové kyseliny MeSH
- krystalografie rentgenová metody MeSH
- molekulární sekvence - údaje MeSH
- roztoky chemie MeSH
- sekvence nukleotidů MeSH
- trifluorethanol chemie MeSH
- voda chemie metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA MeSH
- roztoky MeSH
- trifluorethanol MeSH
- voda MeSH
