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Cruciform extrusion propensity of human translocation-mediating palindromic AT-rich repeats.

Kogo H, Inagaki H, Ohye T, Kato T, Emanuel BS, Kurahashi H - Nucleic Acids Res. (2007)

Bottom Line: The resultant deletions are putatively mediated by central cleavage by the structure-specific endonuclease SbcCD, indicating the possibility of a cruciform conformation in vivo.Insertion of a short spacer at the centre of the PATRR22 greatly reduces both its cruciform extrusion in vitro and instability in vivo.Taken together, cruciform extrusion propensity depends on the length and central symmetry of the PATRR, and is likely to determine the instability that leads to recurrent translocations in humans.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan.

ABSTRACT
There is an emerging consensus that secondary structures of DNA have the potential for genomic instability. Palindromic AT-rich repeats (PATRRs) are a characteristic sequence identified at each breakpoint of the recurrent constitutional t(11;22) and t(17;22) translocations in humans, named PATRR22 (approximately 600 bp), PATRR11 (approximately 450 bp) and PATRR17 (approximately 190 bp). The secondary structure-forming propensity in vitro and the instability in vivo have been experimentally evaluated for various PATRRs that differ regarding their size and symmetry. At physiological ionic strength, a cruciform structure is most frequently observed for the symmetric PATRR22, less often for the symmetric PATRR11, but not for the other PATRRs. In wild-type E. coli, only these two PATRRs undergo extensive instability, consistent with the relatively high incidence of the t(11;22) in humans. The resultant deletions are putatively mediated by central cleavage by the structure-specific endonuclease SbcCD, indicating the possibility of a cruciform conformation in vivo. Insertion of a short spacer at the centre of the PATRR22 greatly reduces both its cruciform extrusion in vitro and instability in vivo. Taken together, cruciform extrusion propensity depends on the length and central symmetry of the PATRR, and is likely to determine the instability that leads to recurrent translocations in humans.

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Effects of salt concentration on cruciform extrusion from various PATRR-containing plasmids. (A) Gel images showing electrophoretic mobility shifts after incubation in TE buffer containing various salt concentrations. Black arrows indicate the positions of supercoiled plasmids. Gray arrows and gray bars indicate the mobility shifts of plasmids due to relaxation by cruciform formation. Fully relaxed forms (gray arrows) were observed in plasmids containing the PATRR11-long (11L), the PATRR22-pal (22P) and the PATRR22-quasi (22Q) and partially relaxed forms (gray bars) were observed in plasmids containing the PATRR11-long (11L), the PATRR11-short (11S) and the PATRR17-long (17L). At relatively low concentrations of NaCl (0–50 mM), most PATRR-containing plasmids, except for the PATRR17-short (17S), exhibited mobility shifts. In contrast, only the plasmids containing the PATRR11-long (11L) and the PATRR22-pal (22P) exhibited significant mobility shifts at relatively physiologic salt concentrations (75–150 mM). The two clusters of bands in the PATRR11-long (11L) exhibit different salt concentration dependency. (B) Quantitative analysis of cruciform-forming ratios in PATRR-containing plasmids incubated at 37°C with various salt concentrations. The band intensities were quantified using NIH image, and the percentages of the shifted bands were plotted against the concentration of NaCl. Data represent mean ± SD of three independent assays.
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Figure 3: Effects of salt concentration on cruciform extrusion from various PATRR-containing plasmids. (A) Gel images showing electrophoretic mobility shifts after incubation in TE buffer containing various salt concentrations. Black arrows indicate the positions of supercoiled plasmids. Gray arrows and gray bars indicate the mobility shifts of plasmids due to relaxation by cruciform formation. Fully relaxed forms (gray arrows) were observed in plasmids containing the PATRR11-long (11L), the PATRR22-pal (22P) and the PATRR22-quasi (22Q) and partially relaxed forms (gray bars) were observed in plasmids containing the PATRR11-long (11L), the PATRR11-short (11S) and the PATRR17-long (17L). At relatively low concentrations of NaCl (0–50 mM), most PATRR-containing plasmids, except for the PATRR17-short (17S), exhibited mobility shifts. In contrast, only the plasmids containing the PATRR11-long (11L) and the PATRR22-pal (22P) exhibited significant mobility shifts at relatively physiologic salt concentrations (75–150 mM). The two clusters of bands in the PATRR11-long (11L) exhibit different salt concentration dependency. (B) Quantitative analysis of cruciform-forming ratios in PATRR-containing plasmids incubated at 37°C with various salt concentrations. The band intensities were quantified using NIH image, and the percentages of the shifted bands were plotted against the concentration of NaCl. Data represent mean ± SD of three independent assays.

Mentions: We analysed the cruciform extrusion propensities of various PATRRs by incubating the plasmids in various salt concentrations at 37°C. Electrophoretic mobility shifts were observed with all PATRR-containing plasmids except for the PATRR17-short when incubated in relatively low salt concentration buffers (0–50 mM NaCl) (Figure 3A). Quantitative analysis of the ratio of the relaxed to the supercoiled plasmid bands demonstrated that secondary structures were formed most efficiently by the PATRR11-long and the PATRR22-pal. Secondary structure is less frequently adopted for the PATRR11-short, the PATRR17-long and the PATRR22-quasi, and not at all by the PATRR17-short (Figure 3B). In contrast, only the PATRR11-long and the PATRR22-pal showed significant cruciform extrusion among various PATRRs in physiologic salt concentrations (75–150 mM NaCl) (Figure 3A). Interestingly, the PATRR22-pal and -quasi exhibited striking difference in their cruciform extrusion abilities at relatively high salt concentrations. This result points out the importance of perfect symmetry at the centre for cruciform extrusion under physiologic conditions. Quantitative analysis at high salt concentration clearly demonstrated that the propensity for cruciform extrusion was highest with the PATRR22-pal, followed by the PATRR11-long and lowest with the PATRR17-long among the three symmetric PATRRs (Figure 3B). This result is quite consistent with previous in silico studies (12), and indicates the striking correlation between cruciform-forming propensity and putative translocation susceptibility in humans.Figure 3.


Cruciform extrusion propensity of human translocation-mediating palindromic AT-rich repeats.

Kogo H, Inagaki H, Ohye T, Kato T, Emanuel BS, Kurahashi H - Nucleic Acids Res. (2007)

Effects of salt concentration on cruciform extrusion from various PATRR-containing plasmids. (A) Gel images showing electrophoretic mobility shifts after incubation in TE buffer containing various salt concentrations. Black arrows indicate the positions of supercoiled plasmids. Gray arrows and gray bars indicate the mobility shifts of plasmids due to relaxation by cruciform formation. Fully relaxed forms (gray arrows) were observed in plasmids containing the PATRR11-long (11L), the PATRR22-pal (22P) and the PATRR22-quasi (22Q) and partially relaxed forms (gray bars) were observed in plasmids containing the PATRR11-long (11L), the PATRR11-short (11S) and the PATRR17-long (17L). At relatively low concentrations of NaCl (0–50 mM), most PATRR-containing plasmids, except for the PATRR17-short (17S), exhibited mobility shifts. In contrast, only the plasmids containing the PATRR11-long (11L) and the PATRR22-pal (22P) exhibited significant mobility shifts at relatively physiologic salt concentrations (75–150 mM). The two clusters of bands in the PATRR11-long (11L) exhibit different salt concentration dependency. (B) Quantitative analysis of cruciform-forming ratios in PATRR-containing plasmids incubated at 37°C with various salt concentrations. The band intensities were quantified using NIH image, and the percentages of the shifted bands were plotted against the concentration of NaCl. Data represent mean ± SD of three independent assays.
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Related In: Results  -  Collection

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Figure 3: Effects of salt concentration on cruciform extrusion from various PATRR-containing plasmids. (A) Gel images showing electrophoretic mobility shifts after incubation in TE buffer containing various salt concentrations. Black arrows indicate the positions of supercoiled plasmids. Gray arrows and gray bars indicate the mobility shifts of plasmids due to relaxation by cruciform formation. Fully relaxed forms (gray arrows) were observed in plasmids containing the PATRR11-long (11L), the PATRR22-pal (22P) and the PATRR22-quasi (22Q) and partially relaxed forms (gray bars) were observed in plasmids containing the PATRR11-long (11L), the PATRR11-short (11S) and the PATRR17-long (17L). At relatively low concentrations of NaCl (0–50 mM), most PATRR-containing plasmids, except for the PATRR17-short (17S), exhibited mobility shifts. In contrast, only the plasmids containing the PATRR11-long (11L) and the PATRR22-pal (22P) exhibited significant mobility shifts at relatively physiologic salt concentrations (75–150 mM). The two clusters of bands in the PATRR11-long (11L) exhibit different salt concentration dependency. (B) Quantitative analysis of cruciform-forming ratios in PATRR-containing plasmids incubated at 37°C with various salt concentrations. The band intensities were quantified using NIH image, and the percentages of the shifted bands were plotted against the concentration of NaCl. Data represent mean ± SD of three independent assays.
Mentions: We analysed the cruciform extrusion propensities of various PATRRs by incubating the plasmids in various salt concentrations at 37°C. Electrophoretic mobility shifts were observed with all PATRR-containing plasmids except for the PATRR17-short when incubated in relatively low salt concentration buffers (0–50 mM NaCl) (Figure 3A). Quantitative analysis of the ratio of the relaxed to the supercoiled plasmid bands demonstrated that secondary structures were formed most efficiently by the PATRR11-long and the PATRR22-pal. Secondary structure is less frequently adopted for the PATRR11-short, the PATRR17-long and the PATRR22-quasi, and not at all by the PATRR17-short (Figure 3B). In contrast, only the PATRR11-long and the PATRR22-pal showed significant cruciform extrusion among various PATRRs in physiologic salt concentrations (75–150 mM NaCl) (Figure 3A). Interestingly, the PATRR22-pal and -quasi exhibited striking difference in their cruciform extrusion abilities at relatively high salt concentrations. This result points out the importance of perfect symmetry at the centre for cruciform extrusion under physiologic conditions. Quantitative analysis at high salt concentration clearly demonstrated that the propensity for cruciform extrusion was highest with the PATRR22-pal, followed by the PATRR11-long and lowest with the PATRR17-long among the three symmetric PATRRs (Figure 3B). This result is quite consistent with previous in silico studies (12), and indicates the striking correlation between cruciform-forming propensity and putative translocation susceptibility in humans.Figure 3.

Bottom Line: The resultant deletions are putatively mediated by central cleavage by the structure-specific endonuclease SbcCD, indicating the possibility of a cruciform conformation in vivo.Insertion of a short spacer at the centre of the PATRR22 greatly reduces both its cruciform extrusion in vitro and instability in vivo.Taken together, cruciform extrusion propensity depends on the length and central symmetry of the PATRR, and is likely to determine the instability that leads to recurrent translocations in humans.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Genetics, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi 470-1192, Japan.

ABSTRACT
There is an emerging consensus that secondary structures of DNA have the potential for genomic instability. Palindromic AT-rich repeats (PATRRs) are a characteristic sequence identified at each breakpoint of the recurrent constitutional t(11;22) and t(17;22) translocations in humans, named PATRR22 (approximately 600 bp), PATRR11 (approximately 450 bp) and PATRR17 (approximately 190 bp). The secondary structure-forming propensity in vitro and the instability in vivo have been experimentally evaluated for various PATRRs that differ regarding their size and symmetry. At physiological ionic strength, a cruciform structure is most frequently observed for the symmetric PATRR22, less often for the symmetric PATRR11, but not for the other PATRRs. In wild-type E. coli, only these two PATRRs undergo extensive instability, consistent with the relatively high incidence of the t(11;22) in humans. The resultant deletions are putatively mediated by central cleavage by the structure-specific endonuclease SbcCD, indicating the possibility of a cruciform conformation in vivo. Insertion of a short spacer at the centre of the PATRR22 greatly reduces both its cruciform extrusion in vitro and instability in vivo. Taken together, cruciform extrusion propensity depends on the length and central symmetry of the PATRR, and is likely to determine the instability that leads to recurrent translocations in humans.

Show MeSH
Related in: MedlinePlus