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Intrastrand triplex DNA repeats in bacteria: a source of genomic instability.

Holder IT, Wagner S, Xiong P, Sinn M, Frickey T, Meyer A, Hartig JS - Nucleic Acids Res. (2015)

Bottom Line: In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse.In conclusion we link intrastrand triplex motifs with the induction of genomic instability.We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany.

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Structural characterization of TM sequences. (A) Circular Dichroism Spectroscopy of TM (TM type A in blue, TM type B in red) and control (containing for mutations, green) oligonucleotides. (B) Imino proton area of 1H-NMR spectra of TM and control oligonucleotides. (C) In vivo DMS probing of the TM sequence found at the genomic site 6′ in comparison to the 6′ control mutant: Primer extension reaction analyzed on a 10% denaturing PAGE. DMS treated probes (DMS) are shown in comparison to non-treated DNA (untreated) and the sequencing reaction for purine sequencing, according to Maxam and Gilbert (52).
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Figure 3: Structural characterization of TM sequences. (A) Circular Dichroism Spectroscopy of TM (TM type A in blue, TM type B in red) and control (containing for mutations, green) oligonucleotides. (B) Imino proton area of 1H-NMR spectra of TM and control oligonucleotides. (C) In vivo DMS probing of the TM sequence found at the genomic site 6′ in comparison to the 6′ control mutant: Primer extension reaction analyzed on a 10% denaturing PAGE. DMS treated probes (DMS) are shown in comparison to non-treated DNA (untreated) and the sequencing reaction for purine sequencing, according to Maxam and Gilbert (52).

Mentions: In order to confirm that the identified TMECO forms a stable triplex structure, oligonucleotides were characterized via CD and NMR spectroscopy. Characteristic CD spectra of DNA triplex structures differ with the sequence of the oligonucleotide (64). However, most of the intramolecular triplex DNA show a minimum around 240 nm, a maximum around 257 nm and a second minimum at ∼280 nm (39,65). These peaks were also observed in the CD spectra of the TMECO oligonucleotides. The consensus sequence of the TMECO found in E. coli K-12 substrain MG1655 is shown in Figure 2A. We measured CD spectra for two types of TMECO: TMECO type A with the sequence 5′-TTA-3′ and TMECO type B with the sequence 5′-CCG-3′ in the second loop. We investigated both TMECO types with and without one mismatched basepair (mm), respectively (Figure 3A). Furthermore we analyzed a control sequence (5′-CCCTCGCCCCTTTGCCGAGAGCGTTAGCGTGAGCGG-3′), which contains four G to C mutations and should not be able to form a triplex—this sequence yielded spectra that resembles duplex (B-DNA) structures (Figure 3A and Supplementary Figure S2E) (64). We found the structures to be very stable as CD spectra showed the characteristic peaks up to a temperature of 75°C, although CD signatures decrease with increasing temperature (Supplementary Figure S2). We compared the influence of magnesium on triplex stability and found only minor changes in CD and thermal denaturation spectra (Supplementary Figure S9) as well as in TMECO CD spectra at different temperatures (Supplementary Figure S10). Next, we determined the stability of the TMECO oligonucleotides (5 μM) by thermal denaturation studies: melting temperatures of 82 ± 4°C, 78 ± 1°C, 74 ± 1°C and 70 ± 1°C were determined for TMECO type A, TMECO type B, TMECO type A mm and TMECO type B mm, respectively (Supplementary Figure S2F). Although CD spectra showed minima and maxima that were observed for triplex structures before, characteristic peaks for parallel G-quadruplex structures are very similar (minimum at 240 nm and maximum at 260 nm). The ability of G-rich triplex sequences to fold into quadruplex structures has been observed before (66). The G-rich part of the TM sequence could in principle form an intramolecular G-quadruplex composed of 3 stacked guanine tetrads (Supplementary Figure S1B) competing with triplex formation. In order to exclude quadruplex formation and prove triplex folding, we carried out NMR measurements. The 1H-NMR spectra of TMECO oligonucleotides displays 18 sharp signals in the imino proton range that clearly demonstrate the formation of well-defined triplex structures (Figure 3B). If an intramolecular G-quadruplex structure would form we would expect much less imino proton signals (3–4 signals). However, when complementary strands were added, CD signals characteristic for duplex structures were observed and the NMR spectrum of TMECO type B mm showed less and broader signals in the imino proton range (Supplementary Figure S3).


Intrastrand triplex DNA repeats in bacteria: a source of genomic instability.

Holder IT, Wagner S, Xiong P, Sinn M, Frickey T, Meyer A, Hartig JS - Nucleic Acids Res. (2015)

Structural characterization of TM sequences. (A) Circular Dichroism Spectroscopy of TM (TM type A in blue, TM type B in red) and control (containing for mutations, green) oligonucleotides. (B) Imino proton area of 1H-NMR spectra of TM and control oligonucleotides. (C) In vivo DMS probing of the TM sequence found at the genomic site 6′ in comparison to the 6′ control mutant: Primer extension reaction analyzed on a 10% denaturing PAGE. DMS treated probes (DMS) are shown in comparison to non-treated DNA (untreated) and the sequencing reaction for purine sequencing, according to Maxam and Gilbert (52).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4666352&req=5

Figure 3: Structural characterization of TM sequences. (A) Circular Dichroism Spectroscopy of TM (TM type A in blue, TM type B in red) and control (containing for mutations, green) oligonucleotides. (B) Imino proton area of 1H-NMR spectra of TM and control oligonucleotides. (C) In vivo DMS probing of the TM sequence found at the genomic site 6′ in comparison to the 6′ control mutant: Primer extension reaction analyzed on a 10% denaturing PAGE. DMS treated probes (DMS) are shown in comparison to non-treated DNA (untreated) and the sequencing reaction for purine sequencing, according to Maxam and Gilbert (52).
Mentions: In order to confirm that the identified TMECO forms a stable triplex structure, oligonucleotides were characterized via CD and NMR spectroscopy. Characteristic CD spectra of DNA triplex structures differ with the sequence of the oligonucleotide (64). However, most of the intramolecular triplex DNA show a minimum around 240 nm, a maximum around 257 nm and a second minimum at ∼280 nm (39,65). These peaks were also observed in the CD spectra of the TMECO oligonucleotides. The consensus sequence of the TMECO found in E. coli K-12 substrain MG1655 is shown in Figure 2A. We measured CD spectra for two types of TMECO: TMECO type A with the sequence 5′-TTA-3′ and TMECO type B with the sequence 5′-CCG-3′ in the second loop. We investigated both TMECO types with and without one mismatched basepair (mm), respectively (Figure 3A). Furthermore we analyzed a control sequence (5′-CCCTCGCCCCTTTGCCGAGAGCGTTAGCGTGAGCGG-3′), which contains four G to C mutations and should not be able to form a triplex—this sequence yielded spectra that resembles duplex (B-DNA) structures (Figure 3A and Supplementary Figure S2E) (64). We found the structures to be very stable as CD spectra showed the characteristic peaks up to a temperature of 75°C, although CD signatures decrease with increasing temperature (Supplementary Figure S2). We compared the influence of magnesium on triplex stability and found only minor changes in CD and thermal denaturation spectra (Supplementary Figure S9) as well as in TMECO CD spectra at different temperatures (Supplementary Figure S10). Next, we determined the stability of the TMECO oligonucleotides (5 μM) by thermal denaturation studies: melting temperatures of 82 ± 4°C, 78 ± 1°C, 74 ± 1°C and 70 ± 1°C were determined for TMECO type A, TMECO type B, TMECO type A mm and TMECO type B mm, respectively (Supplementary Figure S2F). Although CD spectra showed minima and maxima that were observed for triplex structures before, characteristic peaks for parallel G-quadruplex structures are very similar (minimum at 240 nm and maximum at 260 nm). The ability of G-rich triplex sequences to fold into quadruplex structures has been observed before (66). The G-rich part of the TM sequence could in principle form an intramolecular G-quadruplex composed of 3 stacked guanine tetrads (Supplementary Figure S1B) competing with triplex formation. In order to exclude quadruplex formation and prove triplex folding, we carried out NMR measurements. The 1H-NMR spectra of TMECO oligonucleotides displays 18 sharp signals in the imino proton range that clearly demonstrate the formation of well-defined triplex structures (Figure 3B). If an intramolecular G-quadruplex structure would form we would expect much less imino proton signals (3–4 signals). However, when complementary strands were added, CD signals characteristic for duplex structures were observed and the NMR spectrum of TMECO type B mm showed less and broader signals in the imino proton range (Supplementary Figure S3).

Bottom Line: In addition, we found very high frequencies of TM motifs in certain Enterobacteria and Cyanobacteria that were previously described as genetically highly diverse.In conclusion we link intrastrand triplex motifs with the induction of genomic instability.We speculate that the observed instability might be an adaptive feature of these genomes that creates variation for natural selection to act upon.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Konstanz Research School Chemical Biology (KoRS-CB), University of Konstanz, Universitätsstrasse 10, 78457 Konstanz, Germany.

Show MeSH
Related in: MedlinePlus