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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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Deletion of the PATRR22-pal requires SbcC activity. The SbcC-dependency of the PATRR22-pal deletions was evaluated using various E. coli strains. Relevant genotypes for each strain are indicated in parentheses. The insert size of PATRR22s was examined by colony PCR amplification. The bands above the dotted lines represent intact inserts. The PATRR22-pal was extensively deleted in the wild-type strain (AB1157), but was remarkably kept intact in two independent SbcC mutant strains (SURE and CES200). The PATRR22-quasi was stable in all strains examined. Premature termination products of PCR reactions were observed as smaller-sized bands when the template DNA retained its palindromic nature.
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Figure 6: Deletion of the PATRR22-pal requires SbcC activity. The SbcC-dependency of the PATRR22-pal deletions was evaluated using various E. coli strains. Relevant genotypes for each strain are indicated in parentheses. The insert size of PATRR22s was examined by colony PCR amplification. The bands above the dotted lines represent intact inserts. The PATRR22-pal was extensively deleted in the wild-type strain (AB1157), but was remarkably kept intact in two independent SbcC mutant strains (SURE and CES200). The PATRR22-quasi was stable in all strains examined. Premature termination products of PCR reactions were observed as smaller-sized bands when the template DNA retained its palindromic nature.

Mentions: To confirm the SbcC-dependency of the PATRR22-pal deletions, we examined the instability of the PATRR22-pal and -quasi in various E. coli strains by the colony PCR method for rapid and sensitive analysis. The PATRR22-pal were extensively deleted in a wild-type strain (Figure 6, AB1157), but were kept intact in most, but not all, colonies in two independent SbcC mutant strains (Figure 6, SURE and CES200). The other relevant mutations that are in the SURE and CES200 strains, such as recB, recC, recJ and sbcB, did not inhibit the deletions as demonstrated by the use of the other mutant strains (Figure 6, RIK200, JC5519 and BW1012). This result suggests that the hairpin endonuclease activity of SbcCD is crucial for the instability of PATRR sequences, as has been demonstrated for the instability of short artificial palindromes (16). Furthermore, the PATRR22-quasi was not conspicuously deleted in the E. coli strains examined (Figure 6).Figure 6.


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)

Deletion of the PATRR22-pal requires SbcC activity. The SbcC-dependency of the PATRR22-pal deletions was evaluated using various E. coli strains. Relevant genotypes for each strain are indicated in parentheses. The insert size of PATRR22s was examined by colony PCR amplification. The bands above the dotted lines represent intact inserts. The PATRR22-pal was extensively deleted in the wild-type strain (AB1157), but was remarkably kept intact in two independent SbcC mutant strains (SURE and CES200). The PATRR22-quasi was stable in all strains examined. Premature termination products of PCR reactions were observed as smaller-sized bands when the template DNA retained its palindromic nature.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 6: Deletion of the PATRR22-pal requires SbcC activity. The SbcC-dependency of the PATRR22-pal deletions was evaluated using various E. coli strains. Relevant genotypes for each strain are indicated in parentheses. The insert size of PATRR22s was examined by colony PCR amplification. The bands above the dotted lines represent intact inserts. The PATRR22-pal was extensively deleted in the wild-type strain (AB1157), but was remarkably kept intact in two independent SbcC mutant strains (SURE and CES200). The PATRR22-quasi was stable in all strains examined. Premature termination products of PCR reactions were observed as smaller-sized bands when the template DNA retained its palindromic nature.
Mentions: To confirm the SbcC-dependency of the PATRR22-pal deletions, we examined the instability of the PATRR22-pal and -quasi in various E. coli strains by the colony PCR method for rapid and sensitive analysis. The PATRR22-pal were extensively deleted in a wild-type strain (Figure 6, AB1157), but were kept intact in most, but not all, colonies in two independent SbcC mutant strains (Figure 6, SURE and CES200). The other relevant mutations that are in the SURE and CES200 strains, such as recB, recC, recJ and sbcB, did not inhibit the deletions as demonstrated by the use of the other mutant strains (Figure 6, RIK200, JC5519 and BW1012). This result suggests that the hairpin endonuclease activity of SbcCD is crucial for the instability of PATRR sequences, as has been demonstrated for the instability of short artificial palindromes (16). Furthermore, the PATRR22-quasi was not conspicuously deleted in the E. coli strains examined (Figure 6).Figure 6.

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