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The structure of an endogenous Drosophila centromere reveals the prevalence of tandemly repeated sequences able to form i-motifs.

Garavís M, Méndez-Lago M, Gabelica V, Whitehead SL, González C, Villasante A - Sci Rep (2015)

Bottom Line: In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements).Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs.The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049 Madrid, Spain.

ABSTRACT
Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called 'finished' genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.

No MeSH data available.


Related in: MedlinePlus

The centromeric dodeca satellite DNA is able to form dimeric i-motif structures.Imino region of the NMR spectra of the C-rich strands of the undeca (a) and dodeca repeats (b). Experimental conditions: Oligo concentration = 0.8 mM, 25 mM sodium phosphate, 100 mM NaCl, T = 5 °C, pH 4. CD spectra of the C-rich strands of undeca (c) and dodeca repeats (d) at different temperatures. Oligo concentration = 100 μM, same buffer as the NMR experiments. Mass spectrometry data showing the peaks of the single stranded [1] and dimeric [2] species formed by C-rich strands of undeca (e) and dodeca (f). Buffer conditions: 100 mM NH4OAc, spectra at pH 4. See Supplementary Fig. S7 legend for details.
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f3: The centromeric dodeca satellite DNA is able to form dimeric i-motif structures.Imino region of the NMR spectra of the C-rich strands of the undeca (a) and dodeca repeats (b). Experimental conditions: Oligo concentration = 0.8 mM, 25 mM sodium phosphate, 100 mM NaCl, T = 5 °C, pH 4. CD spectra of the C-rich strands of undeca (c) and dodeca repeats (d) at different temperatures. Oligo concentration = 100 μM, same buffer as the NMR experiments. Mass spectrometry data showing the peaks of the single stranded [1] and dimeric [2] species formed by C-rich strands of undeca (e) and dodeca (f). Buffer conditions: 100 mM NH4OAc, spectra at pH 4. See Supplementary Fig. S7 legend for details.

Mentions: To determine the structural behavior of the dodeca satellite DNA, several oligonucleotides containing the dodeca repeat and, its main variant, the undeca repeat were studied by NMR, circular dichroism (CD) and mass spectrometry (MS). The G-rich and the C-rich strands were analyzed under different experimental conditions (Supplementary Fig. S5 and S6). In all cases, the NMR spectra of the G-rich oligonucleotides indicate the formation of G:C base pairs, and no formation of G-tetrads is observed even at high K+ concentrations (Supplementary Fig. S5). This is in agreement with the formation of intramolecular hairpins previously reported47. However, under acidic conditions the NMR spectra of the oligonucleotides corresponding to the C-rich strand of the dodeca and undeca repeats exhibit sharp imino signals around 15–16 ppm, characteristic of i-motif formation (Fig. 3a,b and Supplementary Fig. S6,8). I-motif formation is confirmed by CD spectra, which show the characteristic strong positive band at 285 nm48 (Fig. 3c,d). CD melting experiments show that these structures are quite stable at pH 4.0, with melting temperatures around 42 °C for dodeca and 45 °C for undeca (Supplementary Fig. S7e,f). Mass spectrometry data clearly indicate the formation of dimeric structures at acidic pH (Fig. 3e,f). The peaks corresponding to the dimeric species are not present at neutral pH and become very intense at pH 4 (Supplementary Fig. S7a–d). This pH dependence is consistent with i-motif structures. Interestingly, no tetrameric species are observed in mass spectrometry experiments, indicating that the structures formed by these oligonucleotides are the result of the self-association of two hairpins; similar dimeric structures as those observed in the A and B box of the human alpha satellite2829. To further explore this similarity, we carried out two-dimensional NMR experiments of the dodeca oligonucleotide. Although a full structural determination is beyond the scope of this study, some interesting information can be readily spotted from the NOESY spectra. Each of the six cytosine imino signals (14.5–16.0 ppm) exhibit NOE cross-peaks with two amino protons (Fig. 4a). The presence of only two cross-peaks with cytosine amino protons instead of four (see Fig. 4c) denotes the formation of C:C+ base pairs between equivalent residues in each subunit. Other NOEs characteristic of i-motifs are also observed, such as imino-imino cross-peaks between adjacent C:C+ base pairs (Fig. 4a) or H1’-H1’ contacts between deoxyribose protons (Fig. 4b). According to these experimental data, we suggest a plausible structural model in which two hairpins self-associate in a head-to-head orientation through the formation of six intermolecular C:C+ base pairs (Fig. 4d).


The structure of an endogenous Drosophila centromere reveals the prevalence of tandemly repeated sequences able to form i-motifs.

Garavís M, Méndez-Lago M, Gabelica V, Whitehead SL, González C, Villasante A - Sci Rep (2015)

The centromeric dodeca satellite DNA is able to form dimeric i-motif structures.Imino region of the NMR spectra of the C-rich strands of the undeca (a) and dodeca repeats (b). Experimental conditions: Oligo concentration = 0.8 mM, 25 mM sodium phosphate, 100 mM NaCl, T = 5 °C, pH 4. CD spectra of the C-rich strands of undeca (c) and dodeca repeats (d) at different temperatures. Oligo concentration = 100 μM, same buffer as the NMR experiments. Mass spectrometry data showing the peaks of the single stranded [1] and dimeric [2] species formed by C-rich strands of undeca (e) and dodeca (f). Buffer conditions: 100 mM NH4OAc, spectra at pH 4. See Supplementary Fig. S7 legend for details.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4542561&req=5

f3: The centromeric dodeca satellite DNA is able to form dimeric i-motif structures.Imino region of the NMR spectra of the C-rich strands of the undeca (a) and dodeca repeats (b). Experimental conditions: Oligo concentration = 0.8 mM, 25 mM sodium phosphate, 100 mM NaCl, T = 5 °C, pH 4. CD spectra of the C-rich strands of undeca (c) and dodeca repeats (d) at different temperatures. Oligo concentration = 100 μM, same buffer as the NMR experiments. Mass spectrometry data showing the peaks of the single stranded [1] and dimeric [2] species formed by C-rich strands of undeca (e) and dodeca (f). Buffer conditions: 100 mM NH4OAc, spectra at pH 4. See Supplementary Fig. S7 legend for details.
Mentions: To determine the structural behavior of the dodeca satellite DNA, several oligonucleotides containing the dodeca repeat and, its main variant, the undeca repeat were studied by NMR, circular dichroism (CD) and mass spectrometry (MS). The G-rich and the C-rich strands were analyzed under different experimental conditions (Supplementary Fig. S5 and S6). In all cases, the NMR spectra of the G-rich oligonucleotides indicate the formation of G:C base pairs, and no formation of G-tetrads is observed even at high K+ concentrations (Supplementary Fig. S5). This is in agreement with the formation of intramolecular hairpins previously reported47. However, under acidic conditions the NMR spectra of the oligonucleotides corresponding to the C-rich strand of the dodeca and undeca repeats exhibit sharp imino signals around 15–16 ppm, characteristic of i-motif formation (Fig. 3a,b and Supplementary Fig. S6,8). I-motif formation is confirmed by CD spectra, which show the characteristic strong positive band at 285 nm48 (Fig. 3c,d). CD melting experiments show that these structures are quite stable at pH 4.0, with melting temperatures around 42 °C for dodeca and 45 °C for undeca (Supplementary Fig. S7e,f). Mass spectrometry data clearly indicate the formation of dimeric structures at acidic pH (Fig. 3e,f). The peaks corresponding to the dimeric species are not present at neutral pH and become very intense at pH 4 (Supplementary Fig. S7a–d). This pH dependence is consistent with i-motif structures. Interestingly, no tetrameric species are observed in mass spectrometry experiments, indicating that the structures formed by these oligonucleotides are the result of the self-association of two hairpins; similar dimeric structures as those observed in the A and B box of the human alpha satellite2829. To further explore this similarity, we carried out two-dimensional NMR experiments of the dodeca oligonucleotide. Although a full structural determination is beyond the scope of this study, some interesting information can be readily spotted from the NOESY spectra. Each of the six cytosine imino signals (14.5–16.0 ppm) exhibit NOE cross-peaks with two amino protons (Fig. 4a). The presence of only two cross-peaks with cytosine amino protons instead of four (see Fig. 4c) denotes the formation of C:C+ base pairs between equivalent residues in each subunit. Other NOEs characteristic of i-motifs are also observed, such as imino-imino cross-peaks between adjacent C:C+ base pairs (Fig. 4a) or H1’-H1’ contacts between deoxyribose protons (Fig. 4b). According to these experimental data, we suggest a plausible structural model in which two hairpins self-associate in a head-to-head orientation through the formation of six intermolecular C:C+ base pairs (Fig. 4d).

Bottom Line: In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements).Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs.The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.

View Article: PubMed Central - PubMed

Affiliation: Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Nicolás Cabrera 1, 28049 Madrid, Spain.

ABSTRACT
Centromeres are the chromosomal loci at which spindle microtubules attach to mediate chromosome segregation during mitosis and meiosis. In most eukaryotes, centromeres are made up of highly repetitive DNA sequences (satellite DNA) interspersed with middle repetitive DNA sequences (transposable elements). Despite the efforts to establish complete genomic sequences of eukaryotic organisms, the so-called 'finished' genomes are not actually complete because the centromeres have not been assembled due to the intrinsic difficulties in constructing both physical maps and complete sequence assemblies of long stretches of tandemly repetitive DNA. Here we show the first molecular structure of an endogenous Drosophila centromere and the ability of the C-rich dodeca satellite strand to form dimeric i-motifs. The finding of i-motif structures in simple and complex centromeric satellite DNAs leads us to suggest that these centromeric sequences may have been selected not by their primary sequence but by their ability to form noncanonical secondary structures.

No MeSH data available.


Related in: MedlinePlus