Limits...
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.

Show MeSH

Related in: MedlinePlus

Electrophoresis mobility shifts caused by cruciform extrusion in closed circular plasmids containing PATRR sequences. (A) Schematic representation of the topological change of circular plasmid DNA caused by cruciform formation. Negative supercoiling is relaxed if a cruciform is formed in the supercoiled plasmid DNA. The degree of relaxation is dependent on the length of the extrusion. (B) Diagram of T7 endonuclease I digestion. If the PATRR sequences (thick lines) adopt a cruciform structure, they will be cut into two fragments by a diagonal cleavage with the T7 endonuclease I. (C) PATRR-containing plasmids prepared by the Triton-lysis method were dissolved in TE (pH 7.5) and incubated at room temperature for 16 h (condition 1), or were dissolved in TE with 100 mM NaCl (pH 7.5) on ice without incubation (condition 2) before the electrophoresis in 0.9% agarose gel. Electrophoretic mobility shifts are observed as ladder bands (brackets) with plasmids containing the PATRR11-long (11L), -short (11S) and the PATRR17-long (17L), or observed as a single band (gray arrows) with plasmids containing the PATRR22-pal (22P) and the PATRR22-quasi (22Q). No mobility shift was observed with plasmids containing the PATRR17-short (17S) and non-palindrome sequence (NC). The positions of monomeric and dimeric supercoiled plasmid DNAs are indicated with black arrows. (D) Plasmids dissolved in TE (pH 7.5) and incubated at room temperature for 16 h (condition 1) and those dissolved in NEB2 buffer on ice without incubation (condition 2) were subjected to T7 endonuclease I digestion on ice, and followed by appropriate restriction enzyme digestion. Vectors and intact PATRR-containing inserts are indicated with arrowheads and black brackets, respectively. T7 endonuclease I-digested, PATRR-containing fragments are indicated with gray brackets. The expected sizes of intact fragments (and T7 endonuclease I-digested fragments) are as follows: PATRR11-long, 900 bp (452 and 448 bp); PATRR11-short, 1140 bp (609 and 531 bp); PATRR17-long, 1009 bp (567 and 442 bp); PATRR17-short, 975 bp (554 and 421 bp); PATRR22-pal, 1044 bp (556 and 488 bp) and PATRR22-quasi, 1035 bp (552 and 483 bp).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1851657&req=5

Figure 2: Electrophoresis mobility shifts caused by cruciform extrusion in closed circular plasmids containing PATRR sequences. (A) Schematic representation of the topological change of circular plasmid DNA caused by cruciform formation. Negative supercoiling is relaxed if a cruciform is formed in the supercoiled plasmid DNA. The degree of relaxation is dependent on the length of the extrusion. (B) Diagram of T7 endonuclease I digestion. If the PATRR sequences (thick lines) adopt a cruciform structure, they will be cut into two fragments by a diagonal cleavage with the T7 endonuclease I. (C) PATRR-containing plasmids prepared by the Triton-lysis method were dissolved in TE (pH 7.5) and incubated at room temperature for 16 h (condition 1), or were dissolved in TE with 100 mM NaCl (pH 7.5) on ice without incubation (condition 2) before the electrophoresis in 0.9% agarose gel. Electrophoretic mobility shifts are observed as ladder bands (brackets) with plasmids containing the PATRR11-long (11L), -short (11S) and the PATRR17-long (17L), or observed as a single band (gray arrows) with plasmids containing the PATRR22-pal (22P) and the PATRR22-quasi (22Q). No mobility shift was observed with plasmids containing the PATRR17-short (17S) and non-palindrome sequence (NC). The positions of monomeric and dimeric supercoiled plasmid DNAs are indicated with black arrows. (D) Plasmids dissolved in TE (pH 7.5) and incubated at room temperature for 16 h (condition 1) and those dissolved in NEB2 buffer on ice without incubation (condition 2) were subjected to T7 endonuclease I digestion on ice, and followed by appropriate restriction enzyme digestion. Vectors and intact PATRR-containing inserts are indicated with arrowheads and black brackets, respectively. T7 endonuclease I-digested, PATRR-containing fragments are indicated with gray brackets. The expected sizes of intact fragments (and T7 endonuclease I-digested fragments) are as follows: PATRR11-long, 900 bp (452 and 448 bp); PATRR11-short, 1140 bp (609 and 531 bp); PATRR17-long, 1009 bp (567 and 442 bp); PATRR17-short, 975 bp (554 and 421 bp); PATRR22-pal, 1044 bp (556 and 488 bp) and PATRR22-quasi, 1035 bp (552 and 483 bp).

Mentions: Palindrome-containing supercoiled plasmid is known to be relaxed when a cruciform is formed by the palindromic DNA sequence (Figure 2A). Consistent with that finding, we previously demonstrated that the PATRR11-short-containing plasmid undergoes a conformational change, which results in a mobility shift on agarose gel electrophoresis (17). We employed a non-denaturing, Triton-lysis method for the preparation of cruciform-free plasmids. The plasmids prepared using this technique are mostly supercoiled upon preparation (Figure 2C, condition 2, black arrows). After incubation at room temperature for 16 h, all of the PATRR-containing plasmids, except for the PATRR17-short, exhibited the following electrophoretic mobility shifts (Figure 2C, condition 1). With plasmids containing PATRR11-long and -short, the shifted bands are seen as a ladder and a smear, which reflect the various linking numbers of the plasmids (Figure 2C, gray brackets). A partial mobility shift of the PATRR17-long plasmid was evident only with the dimeric plasmid (Figure 2C, 17L, condition 1). In contrast to the results seen for the PATRR11s and the PATRR17-long, the shifted bands for the PATRR22-pal and -quasi plasmids appear as single bands migrating at the position of the open circular form (Figure 2C, gray arrows), indicating that the cruciform-forming palindrome sequence is long enough to fully relax the plasmids. Theoretically, transition of about 300 bp of the inverted repeat sequence into a cruciform structure would be sufficient for full relaxation of the ∼4 kb plasmids, which contain, in general, 15–25 superhelical turns in E. coli. This is because one superhelical turn is relaxed for every 10.5 bp of DNA that adopts a cruciform configuration (19). Two clusters of shifted bands are observed in the PATRR11-long plasmids (Figure 2C, 11L, condition 1). They are likely to represent two different stable conformations on the basis of previous observations using two-dimensional gel electrophoresis that demonstrated multiple configurations adopted by the PATRR11-short plasmid (17). Indeed, there is no sequence difference of the PATRRs between the two band clusters (data not shown). In contrast, no mobility shift was observed in a plasmid with non-palindromic sequence (Figure 2C, NC).Figure 2.


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)

Electrophoresis mobility shifts caused by cruciform extrusion in closed circular plasmids containing PATRR sequences. (A) Schematic representation of the topological change of circular plasmid DNA caused by cruciform formation. Negative supercoiling is relaxed if a cruciform is formed in the supercoiled plasmid DNA. The degree of relaxation is dependent on the length of the extrusion. (B) Diagram of T7 endonuclease I digestion. If the PATRR sequences (thick lines) adopt a cruciform structure, they will be cut into two fragments by a diagonal cleavage with the T7 endonuclease I. (C) PATRR-containing plasmids prepared by the Triton-lysis method were dissolved in TE (pH 7.5) and incubated at room temperature for 16 h (condition 1), or were dissolved in TE with 100 mM NaCl (pH 7.5) on ice without incubation (condition 2) before the electrophoresis in 0.9% agarose gel. Electrophoretic mobility shifts are observed as ladder bands (brackets) with plasmids containing the PATRR11-long (11L), -short (11S) and the PATRR17-long (17L), or observed as a single band (gray arrows) with plasmids containing the PATRR22-pal (22P) and the PATRR22-quasi (22Q). No mobility shift was observed with plasmids containing the PATRR17-short (17S) and non-palindrome sequence (NC). The positions of monomeric and dimeric supercoiled plasmid DNAs are indicated with black arrows. (D) Plasmids dissolved in TE (pH 7.5) and incubated at room temperature for 16 h (condition 1) and those dissolved in NEB2 buffer on ice without incubation (condition 2) were subjected to T7 endonuclease I digestion on ice, and followed by appropriate restriction enzyme digestion. Vectors and intact PATRR-containing inserts are indicated with arrowheads and black brackets, respectively. T7 endonuclease I-digested, PATRR-containing fragments are indicated with gray brackets. The expected sizes of intact fragments (and T7 endonuclease I-digested fragments) are as follows: PATRR11-long, 900 bp (452 and 448 bp); PATRR11-short, 1140 bp (609 and 531 bp); PATRR17-long, 1009 bp (567 and 442 bp); PATRR17-short, 975 bp (554 and 421 bp); PATRR22-pal, 1044 bp (556 and 488 bp) and PATRR22-quasi, 1035 bp (552 and 483 bp).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 2: Electrophoresis mobility shifts caused by cruciform extrusion in closed circular plasmids containing PATRR sequences. (A) Schematic representation of the topological change of circular plasmid DNA caused by cruciform formation. Negative supercoiling is relaxed if a cruciform is formed in the supercoiled plasmid DNA. The degree of relaxation is dependent on the length of the extrusion. (B) Diagram of T7 endonuclease I digestion. If the PATRR sequences (thick lines) adopt a cruciform structure, they will be cut into two fragments by a diagonal cleavage with the T7 endonuclease I. (C) PATRR-containing plasmids prepared by the Triton-lysis method were dissolved in TE (pH 7.5) and incubated at room temperature for 16 h (condition 1), or were dissolved in TE with 100 mM NaCl (pH 7.5) on ice without incubation (condition 2) before the electrophoresis in 0.9% agarose gel. Electrophoretic mobility shifts are observed as ladder bands (brackets) with plasmids containing the PATRR11-long (11L), -short (11S) and the PATRR17-long (17L), or observed as a single band (gray arrows) with plasmids containing the PATRR22-pal (22P) and the PATRR22-quasi (22Q). No mobility shift was observed with plasmids containing the PATRR17-short (17S) and non-palindrome sequence (NC). The positions of monomeric and dimeric supercoiled plasmid DNAs are indicated with black arrows. (D) Plasmids dissolved in TE (pH 7.5) and incubated at room temperature for 16 h (condition 1) and those dissolved in NEB2 buffer on ice without incubation (condition 2) were subjected to T7 endonuclease I digestion on ice, and followed by appropriate restriction enzyme digestion. Vectors and intact PATRR-containing inserts are indicated with arrowheads and black brackets, respectively. T7 endonuclease I-digested, PATRR-containing fragments are indicated with gray brackets. The expected sizes of intact fragments (and T7 endonuclease I-digested fragments) are as follows: PATRR11-long, 900 bp (452 and 448 bp); PATRR11-short, 1140 bp (609 and 531 bp); PATRR17-long, 1009 bp (567 and 442 bp); PATRR17-short, 975 bp (554 and 421 bp); PATRR22-pal, 1044 bp (556 and 488 bp) and PATRR22-quasi, 1035 bp (552 and 483 bp).
Mentions: Palindrome-containing supercoiled plasmid is known to be relaxed when a cruciform is formed by the palindromic DNA sequence (Figure 2A). Consistent with that finding, we previously demonstrated that the PATRR11-short-containing plasmid undergoes a conformational change, which results in a mobility shift on agarose gel electrophoresis (17). We employed a non-denaturing, Triton-lysis method for the preparation of cruciform-free plasmids. The plasmids prepared using this technique are mostly supercoiled upon preparation (Figure 2C, condition 2, black arrows). After incubation at room temperature for 16 h, all of the PATRR-containing plasmids, except for the PATRR17-short, exhibited the following electrophoretic mobility shifts (Figure 2C, condition 1). With plasmids containing PATRR11-long and -short, the shifted bands are seen as a ladder and a smear, which reflect the various linking numbers of the plasmids (Figure 2C, gray brackets). A partial mobility shift of the PATRR17-long plasmid was evident only with the dimeric plasmid (Figure 2C, 17L, condition 1). In contrast to the results seen for the PATRR11s and the PATRR17-long, the shifted bands for the PATRR22-pal and -quasi plasmids appear as single bands migrating at the position of the open circular form (Figure 2C, gray arrows), indicating that the cruciform-forming palindrome sequence is long enough to fully relax the plasmids. Theoretically, transition of about 300 bp of the inverted repeat sequence into a cruciform structure would be sufficient for full relaxation of the ∼4 kb plasmids, which contain, in general, 15–25 superhelical turns in E. coli. This is because one superhelical turn is relaxed for every 10.5 bp of DNA that adopts a cruciform configuration (19). Two clusters of shifted bands are observed in the PATRR11-long plasmids (Figure 2C, 11L, condition 1). They are likely to represent two different stable conformations on the basis of previous observations using two-dimensional gel electrophoresis that demonstrated multiple configurations adopted by the PATRR11-short plasmid (17). Indeed, there is no sequence difference of the PATRRs between the two band clusters (data not shown). In contrast, no mobility shift was observed in a plasmid with non-palindromic sequence (Figure 2C, NC).Figure 2.

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