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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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Instabilities of PATRR-containing plasmids in E. coli. The expected fragment sizes containing the intact PATRR sequences are as follows: PATRR11-long, 900 bp; PATRR11-short, 1140 bp; PATRR17-long, 1093 bp; PATRR17-short, 1067 bp; PATRR22-pal, 1044 bp and PATRR22-quasi, 1035 bp. The positions of the intact bands are indicated with black arrowheads. Twelve randomly selected independent clones are represented for each plasmid and each strain. In both SURE and AB1157 strain, the PATRR11-long (11L) and the PATRR17-long (17L) exhibited the same-sized deletions of ∼150 and ∼100 bp, respectively (gray arrowheads). Prominent deletions of various sizes occurred in plasmids containing the PATRR11-long (11L) and the PATRR22-pal (22P) specifically in AB1157 strain (gray bars). The remarkable instability is consistent with their cruciform-forming propensity at physiological conditions in vitro.
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Figure 4: Instabilities of PATRR-containing plasmids in E. coli. The expected fragment sizes containing the intact PATRR sequences are as follows: PATRR11-long, 900 bp; PATRR11-short, 1140 bp; PATRR17-long, 1093 bp; PATRR17-short, 1067 bp; PATRR22-pal, 1044 bp and PATRR22-quasi, 1035 bp. The positions of the intact bands are indicated with black arrowheads. Twelve randomly selected independent clones are represented for each plasmid and each strain. In both SURE and AB1157 strain, the PATRR11-long (11L) and the PATRR17-long (17L) exhibited the same-sized deletions of ∼150 and ∼100 bp, respectively (gray arrowheads). Prominent deletions of various sizes occurred in plasmids containing the PATRR11-long (11L) and the PATRR22-pal (22P) specifically in AB1157 strain (gray bars). The remarkable instability is consistent with their cruciform-forming propensity at physiological conditions in vitro.

Mentions: We first evaluated SbcCD-independent deletion in the SURE strain, which has a mutation in the SbcC gene. When multiple independent clones were examined, PATRR11-long and PATRR17-long underwent partial, short deletions of the same length in the SURE strain (Figure 4, gray arrowheads), whereas other PATRRs were stable. We then analysed the replication direction dependency of these deletions by comparing two plasmids with the PATRR17-long sequence inserted in opposite directions (Figure 5, 17L-p and 17L-d). Similar deletions were observed with the two constructs (data not shown). Sequence analysis of the deletion products identified two types of mirror image rearrangement with respect to the direction of replication that were mediated by a direct repeat consisting of 11 nt (Figure 5). The frequency of the two deletions was clearly biased by the replication direction of the plasmids. The location of the direct repeats was consistent with preferential occurrence of the slippage on the lagging strand based on the knowledge that the ‘donor repeat’ (Figure 5, blue arrowheads) is located closer to the centre than the ‘target repeat’ (Figure 5, black arrowheads) for a slippage (9). These results indicate that deletion of the PATRR17-long and perhaps the PATRR11-long are potentially mediated by replication slipped mispairing at the hairpin structure formed on the lagging strand template.Figure 4.


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)

Instabilities of PATRR-containing plasmids in E. coli. The expected fragment sizes containing the intact PATRR sequences are as follows: PATRR11-long, 900 bp; PATRR11-short, 1140 bp; PATRR17-long, 1093 bp; PATRR17-short, 1067 bp; PATRR22-pal, 1044 bp and PATRR22-quasi, 1035 bp. The positions of the intact bands are indicated with black arrowheads. Twelve randomly selected independent clones are represented for each plasmid and each strain. In both SURE and AB1157 strain, the PATRR11-long (11L) and the PATRR17-long (17L) exhibited the same-sized deletions of ∼150 and ∼100 bp, respectively (gray arrowheads). Prominent deletions of various sizes occurred in plasmids containing the PATRR11-long (11L) and the PATRR22-pal (22P) specifically in AB1157 strain (gray bars). The remarkable instability is consistent with their cruciform-forming propensity at physiological conditions in vitro.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1851657&req=5

Figure 4: Instabilities of PATRR-containing plasmids in E. coli. The expected fragment sizes containing the intact PATRR sequences are as follows: PATRR11-long, 900 bp; PATRR11-short, 1140 bp; PATRR17-long, 1093 bp; PATRR17-short, 1067 bp; PATRR22-pal, 1044 bp and PATRR22-quasi, 1035 bp. The positions of the intact bands are indicated with black arrowheads. Twelve randomly selected independent clones are represented for each plasmid and each strain. In both SURE and AB1157 strain, the PATRR11-long (11L) and the PATRR17-long (17L) exhibited the same-sized deletions of ∼150 and ∼100 bp, respectively (gray arrowheads). Prominent deletions of various sizes occurred in plasmids containing the PATRR11-long (11L) and the PATRR22-pal (22P) specifically in AB1157 strain (gray bars). The remarkable instability is consistent with their cruciform-forming propensity at physiological conditions in vitro.
Mentions: We first evaluated SbcCD-independent deletion in the SURE strain, which has a mutation in the SbcC gene. When multiple independent clones were examined, PATRR11-long and PATRR17-long underwent partial, short deletions of the same length in the SURE strain (Figure 4, gray arrowheads), whereas other PATRRs were stable. We then analysed the replication direction dependency of these deletions by comparing two plasmids with the PATRR17-long sequence inserted in opposite directions (Figure 5, 17L-p and 17L-d). Similar deletions were observed with the two constructs (data not shown). Sequence analysis of the deletion products identified two types of mirror image rearrangement with respect to the direction of replication that were mediated by a direct repeat consisting of 11 nt (Figure 5). The frequency of the two deletions was clearly biased by the replication direction of the plasmids. The location of the direct repeats was consistent with preferential occurrence of the slippage on the lagging strand based on the knowledge that the ‘donor repeat’ (Figure 5, blue arrowheads) is located closer to the centre than the ‘target repeat’ (Figure 5, black arrowheads) for a slippage (9). These results indicate that deletion of the PATRR17-long and perhaps the PATRR11-long are potentially mediated by replication slipped mispairing at the hairpin structure formed on the lagging strand template.Figure 4.

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