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A minimal i-motif stabilized by minor groove G:T:G:T tetrads.

Escaja N, Viladoms J, Garavís M, Villasante A, Pedroso E, González C - Nucleic Acids Res. (2012)

Bottom Line: The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes.This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines.Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

View Article: PubMed Central - PubMed

Affiliation: Departament de Química Orgànica and IBUB, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain.

ABSTRACT
The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

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Regions of the NOESY spectra (tm = 300 ms) of d<pTCGTTTCGTT> in H2O/D2O 9:1. Top: imino-sugar protons region showing cross-peaks involving guanine (G3) and thymine (T1) residues forming G:T base pair (left) and Ar-sugar protons region showing the sequential connectivities T1→C2→G3→T4 (right). Bottom: imino region of protonated cytosine (C2) forming hemi-protonated C:C+ base pair (left) and imino and amino region showing connectivities involving imino protons of thymine (T1) and guanine (G3) residues and amino protons of guanine (G3) and cytosine (C2) residues (right). Conditions: 0.5 mM oligonucleotide concentration, 25 mM phosphate buffer, 100 mM NaCl, T = 5°C, pH 5.1. Cross-peaks of d<pTCGTTTCGTT> are labeled according to the spin systems numbering shown in Figure 1.
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gks911-F4: Regions of the NOESY spectra (tm = 300 ms) of d<pTCGTTTCGTT> in H2O/D2O 9:1. Top: imino-sugar protons region showing cross-peaks involving guanine (G3) and thymine (T1) residues forming G:T base pair (left) and Ar-sugar protons region showing the sequential connectivities T1→C2→G3→T4 (right). Bottom: imino region of protonated cytosine (C2) forming hemi-protonated C:C+ base pair (left) and imino and amino region showing connectivities involving imino protons of thymine (T1) and guanine (G3) residues and amino protons of guanine (G3) and cytosine (C2) residues (right). Conditions: 0.5 mM oligonucleotide concentration, 25 mM phosphate buffer, 100 mM NaCl, T = 5°C, pH 5.1. Cross-peaks of d<pTCGTTTCGTT> are labeled according to the spin systems numbering shown in Figure 1.

Mentions: In the case of the cyclic decamer, the sequence is repetitive and only signals corresponding to five distinct residues are expected for a monomeric species. Interestingly, the number of signals in the NMR spectra of the high concentration species of d<pTCGTTTCGTT> also corresponds with 5 nt. This fact clearly indicates that its structure is a homodimer with a 2-fold symmetry. The NMR spectra of d(TCGTTTCGT) exhibit very similar features. However, this sequence is not completely repetitive, and NMR signals are not degenerated (Figure 4 and Supplementary Figures S5 and S7). Complete spectral assignment in this case is very problematical and, consequently, we decided to focus on the structural determination of the cyclic analog.Figure 4.


A minimal i-motif stabilized by minor groove G:T:G:T tetrads.

Escaja N, Viladoms J, Garavís M, Villasante A, Pedroso E, González C - Nucleic Acids Res. (2012)

Regions of the NOESY spectra (tm = 300 ms) of d<pTCGTTTCGTT> in H2O/D2O 9:1. Top: imino-sugar protons region showing cross-peaks involving guanine (G3) and thymine (T1) residues forming G:T base pair (left) and Ar-sugar protons region showing the sequential connectivities T1→C2→G3→T4 (right). Bottom: imino region of protonated cytosine (C2) forming hemi-protonated C:C+ base pair (left) and imino and amino region showing connectivities involving imino protons of thymine (T1) and guanine (G3) residues and amino protons of guanine (G3) and cytosine (C2) residues (right). Conditions: 0.5 mM oligonucleotide concentration, 25 mM phosphate buffer, 100 mM NaCl, T = 5°C, pH 5.1. Cross-peaks of d<pTCGTTTCGTT> are labeled according to the spin systems numbering shown in Figure 1.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3526289&req=5

gks911-F4: Regions of the NOESY spectra (tm = 300 ms) of d<pTCGTTTCGTT> in H2O/D2O 9:1. Top: imino-sugar protons region showing cross-peaks involving guanine (G3) and thymine (T1) residues forming G:T base pair (left) and Ar-sugar protons region showing the sequential connectivities T1→C2→G3→T4 (right). Bottom: imino region of protonated cytosine (C2) forming hemi-protonated C:C+ base pair (left) and imino and amino region showing connectivities involving imino protons of thymine (T1) and guanine (G3) residues and amino protons of guanine (G3) and cytosine (C2) residues (right). Conditions: 0.5 mM oligonucleotide concentration, 25 mM phosphate buffer, 100 mM NaCl, T = 5°C, pH 5.1. Cross-peaks of d<pTCGTTTCGTT> are labeled according to the spin systems numbering shown in Figure 1.
Mentions: In the case of the cyclic decamer, the sequence is repetitive and only signals corresponding to five distinct residues are expected for a monomeric species. Interestingly, the number of signals in the NMR spectra of the high concentration species of d<pTCGTTTCGTT> also corresponds with 5 nt. This fact clearly indicates that its structure is a homodimer with a 2-fold symmetry. The NMR spectra of d(TCGTTTCGT) exhibit very similar features. However, this sequence is not completely repetitive, and NMR signals are not degenerated (Figure 4 and Supplementary Figures S5 and S7). Complete spectral assignment in this case is very problematical and, consequently, we decided to focus on the structural determination of the cyclic analog.Figure 4.

Bottom Line: The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes.This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines.Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

View Article: PubMed Central - PubMed

Affiliation: Departament de Química Orgànica and IBUB, Universitat de Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain.

ABSTRACT
The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d. Both sequences form dimeric quadruplex structures consisting of a minimal i-motif capped, at both ends, by a slipped minor groove-aligned G:T:G:T tetrad. These mini i-motifs, which do not exhibit the characteristic CD spectra of other i-motif structures, can be observed at neutral pH, although they are more stable under acidic conditions. This finding is particularly relevant since these oligonucleotide sequences do not contain contiguous cytosines. Importantly, these structures resemble the loop moiety adopted by an 11-nucleotide fragment of the conserved centromeric protein B (CENP-B) box motif, which is the binding site for the CENP-B.

Show MeSH
Related in: MedlinePlus