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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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Sequence analysis of the PATRR22-pal deletions in AB1157 strain. The head-to-head arrows represent the palindromic sequence of the PATRR22-pal. Numbers with a plus (+1 to +301) indicate the position of bases from the end of the palindrome (vertical bars) in both the proximal and distal arms, while numbers with a minus indicate the positions of bases outside the PATRR. The larger plus numbers indicate positions closer to the centre (i.e. the palindrome is labelled from +1 to +301 and back to +1). Thin arrows represent the direction of lagging strand synthesis in the PATRR22-pal-pBluescript II-KS (22P-d) and -SK (22P-p) plasmids. Deletion types are classified by the positions of direct repeat pairs (arrowheads) that flank the deleted sequences (dashed lines). One of a pair is located near the centre in the type A deletions, and is located at the distal and proximal ends in the type B and type C deletions, respectively. The different distribution of direct repeats supports the existence of different putative mechanisms for these deletions.
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Figure 7: Sequence analysis of the PATRR22-pal deletions in AB1157 strain. The head-to-head arrows represent the palindromic sequence of the PATRR22-pal. Numbers with a plus (+1 to +301) indicate the position of bases from the end of the palindrome (vertical bars) in both the proximal and distal arms, while numbers with a minus indicate the positions of bases outside the PATRR. The larger plus numbers indicate positions closer to the centre (i.e. the palindrome is labelled from +1 to +301 and back to +1). Thin arrows represent the direction of lagging strand synthesis in the PATRR22-pal-pBluescript II-KS (22P-d) and -SK (22P-p) plasmids. Deletion types are classified by the positions of direct repeat pairs (arrowheads) that flank the deleted sequences (dashed lines). One of a pair is located near the centre in the type A deletions, and is located at the distal and proximal ends in the type B and type C deletions, respectively. The different distribution of direct repeats supports the existence of different putative mechanisms for these deletions.

Mentions: To further characterize the SbcCD-dependent deletions of the PATRR22-pal, the influence of replication direction was analysed using two plasmids with opposite replication directions in the AB1157 strain. As a result, we found that rearranged products could be largely divided into two types; one is significantly dependent on replication direction (Figure 7, types B and C) and the other is relatively independent (Figure 7, type A). Replication direction-dependency for the SbcCD-dependent deletions was rather unexpected, as a previous study demonstrated that such deletions were independent of replication direction (16). The most frequent deletions among types B and C were mediated by the same direct repeats in opposite directions (Figure 7, B-1 and C-1). In all the types B and C deletions, the putative donor repeats on the lagging strand were located around the start of the palindromic sequence, and were more internal than the target repeats (Figure 7, B and C series). These results indicate that types B and C deletions are likely to be mediated by a slipped-replication-mispairing mechanism. In contrast to types B and C deletions, type A deletions were relatively independent of replication direction, and occurred more asymmetrically, i.e. near the centre and the outside of the palindrome (Figure 7, type A). The localization of one deletion end near the centre of the palindrome is in agreement with putative DNA cleavage at the tips of the cruciform structure by the SbcCD endonuclease followed by asymmetric resection of the DNA ends (16). Thus, the type A deletion is likely to be caused by cleavage of the cruciform structure followed by a single-strand annealing mechanism. The assumption of an essential difference between the two types of deletions would be further supported by differences in the direct repeat lengths; the type A deletions utilized longer direct repeats (6–23 bp) than the types B and C deletions (2–7 bp). The length of the direct repeats mediating the latter deletions was consistent with a previous report showing the utilization of short direct repeats (3–8 bp) for replication slippage (20). Although direct evaluation was impossible, the PATRR11-long may also form a cruciform structure in vivo that is responsible for its SbcCD-dependent deletion in wild-type E. coli. Taken together, these results provide the first experimental evidence for cruciform structure formation by long, symmetric PATRR sequences in vivo. This cruciform structure was extremely unstable in wild-type E. coli because of its susceptibility to structure-specific nuclease.Figure 7.


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)

Sequence analysis of the PATRR22-pal deletions in AB1157 strain. The head-to-head arrows represent the palindromic sequence of the PATRR22-pal. Numbers with a plus (+1 to +301) indicate the position of bases from the end of the palindrome (vertical bars) in both the proximal and distal arms, while numbers with a minus indicate the positions of bases outside the PATRR. The larger plus numbers indicate positions closer to the centre (i.e. the palindrome is labelled from +1 to +301 and back to +1). Thin arrows represent the direction of lagging strand synthesis in the PATRR22-pal-pBluescript II-KS (22P-d) and -SK (22P-p) plasmids. Deletion types are classified by the positions of direct repeat pairs (arrowheads) that flank the deleted sequences (dashed lines). One of a pair is located near the centre in the type A deletions, and is located at the distal and proximal ends in the type B and type C deletions, respectively. The different distribution of direct repeats supports the existence of different putative mechanisms for these deletions.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

Figure 7: Sequence analysis of the PATRR22-pal deletions in AB1157 strain. The head-to-head arrows represent the palindromic sequence of the PATRR22-pal. Numbers with a plus (+1 to +301) indicate the position of bases from the end of the palindrome (vertical bars) in both the proximal and distal arms, while numbers with a minus indicate the positions of bases outside the PATRR. The larger plus numbers indicate positions closer to the centre (i.e. the palindrome is labelled from +1 to +301 and back to +1). Thin arrows represent the direction of lagging strand synthesis in the PATRR22-pal-pBluescript II-KS (22P-d) and -SK (22P-p) plasmids. Deletion types are classified by the positions of direct repeat pairs (arrowheads) that flank the deleted sequences (dashed lines). One of a pair is located near the centre in the type A deletions, and is located at the distal and proximal ends in the type B and type C deletions, respectively. The different distribution of direct repeats supports the existence of different putative mechanisms for these deletions.
Mentions: To further characterize the SbcCD-dependent deletions of the PATRR22-pal, the influence of replication direction was analysed using two plasmids with opposite replication directions in the AB1157 strain. As a result, we found that rearranged products could be largely divided into two types; one is significantly dependent on replication direction (Figure 7, types B and C) and the other is relatively independent (Figure 7, type A). Replication direction-dependency for the SbcCD-dependent deletions was rather unexpected, as a previous study demonstrated that such deletions were independent of replication direction (16). The most frequent deletions among types B and C were mediated by the same direct repeats in opposite directions (Figure 7, B-1 and C-1). In all the types B and C deletions, the putative donor repeats on the lagging strand were located around the start of the palindromic sequence, and were more internal than the target repeats (Figure 7, B and C series). These results indicate that types B and C deletions are likely to be mediated by a slipped-replication-mispairing mechanism. In contrast to types B and C deletions, type A deletions were relatively independent of replication direction, and occurred more asymmetrically, i.e. near the centre and the outside of the palindrome (Figure 7, type A). The localization of one deletion end near the centre of the palindrome is in agreement with putative DNA cleavage at the tips of the cruciform structure by the SbcCD endonuclease followed by asymmetric resection of the DNA ends (16). Thus, the type A deletion is likely to be caused by cleavage of the cruciform structure followed by a single-strand annealing mechanism. The assumption of an essential difference between the two types of deletions would be further supported by differences in the direct repeat lengths; the type A deletions utilized longer direct repeats (6–23 bp) than the types B and C deletions (2–7 bp). The length of the direct repeats mediating the latter deletions was consistent with a previous report showing the utilization of short direct repeats (3–8 bp) for replication slippage (20). Although direct evaluation was impossible, the PATRR11-long may also form a cruciform structure in vivo that is responsible for its SbcCD-dependent deletion in wild-type E. coli. Taken together, these results provide the first experimental evidence for cruciform structure formation by long, symmetric PATRR sequences in vivo. This cruciform structure was extremely unstable in wild-type E. coli because of its susceptibility to structure-specific nuclease.Figure 7.

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