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Double-strand breaks in the myotonic dystrophy type 1 and the fragile X syndrome triplet repeat sequences induce different types of mutations in DNA flanking sequences in Escherichia coli.

Kosmider B, Wells RD - Nucleic Acids Res. (2006)

Bottom Line: DSBs at TRS junctions with the vector generated a large number of mutagenic events in flanking sequences whereas DSBs within the repeats elicited no similar products.Surprisingly, DNA sequence analyses on mutant clones revealed the presence of only single deletions of 0.4-1.6 kb including the TRS and the flanking sequence from plasmids originally containing (CGG*CCG)43 but single, double and multiple deletions as well as insertions were found for plasmids originally containing (CTG*CAG)n (where n = 43 or 70).Non-B DNA structures (slipped structures with loops, cruciforms, triplexes and tetraplexes) as well as microhomologies are postulated to participate in the recombination and/or repair processes.

View Article: PubMed Central - PubMed

Affiliation: Center for Genome Research, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Texas Medical Center, 2121 W. Holcombe Boulevard, Houston, TX 77030-3303, USA.

ABSTRACT
The putative role of double-strand breaks (DSBs) created in vitro by restriction enzyme cleavage in or near CGG*CCG or CTG*CAG repeat tracts on their genetic instabilities, both within the repeats and in their flanking sequences, was investigated in an Escherichia coli plasmid system. DSBs at TRS junctions with the vector generated a large number of mutagenic events in flanking sequences whereas DSBs within the repeats elicited no similar products. A substantial enhancement in the number of mutants was caused by transcription of the repeats and by the absence of recombination functions (recA-, recBC-). Surprisingly, DNA sequence analyses on mutant clones revealed the presence of only single deletions of 0.4-1.6 kb including the TRS and the flanking sequence from plasmids originally containing (CGG*CCG)43 but single, double and multiple deletions as well as insertions were found for plasmids originally containing (CTG*CAG)n (where n = 43 or 70). Non-B DNA structures (slipped structures with loops, cruciforms, triplexes and tetraplexes) as well as microhomologies are postulated to participate in the recombination and/or repair processes.

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Influence of the type and orientation of TRS on mutagenic patterns of DSB repair. The percentages of DSB repair products involving single, double and multiple deletions as well as inversions identified in the mutant clones are shown for plasmids harboring: (A) (CGG·CCG)43, in (B) (CTG·CAG)43 and in (C) (CTG·CAG)70. The orientations of the TRSs were explained in the legend to Figure 1. White bars indicate orientation I, whereas filled bars show orientation II. The very small percentages near zero are actually zero but are shown as larger values on this Figure to enable visualization. The data for this Figure was derived from Figure 2. Percentages are shown for each type of product for a given orientation.
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fig3: Influence of the type and orientation of TRS on mutagenic patterns of DSB repair. The percentages of DSB repair products involving single, double and multiple deletions as well as inversions identified in the mutant clones are shown for plasmids harboring: (A) (CGG·CCG)43, in (B) (CTG·CAG)43 and in (C) (CTG·CAG)70. The orientations of the TRSs were explained in the legend to Figure 1. White bars indicate orientation I, whereas filled bars show orientation II. The very small percentages near zero are actually zero but are shown as larger values on this Figure to enable visualization. The data for this Figure was derived from Figure 2. Percentages are shown for each type of product for a given orientation.

Mentions: In summary, we found an influence of the type, orientation and the length of the repeat tracts on the induction of the mutagenic events (Figure 3). For (CGG•CCG)43, only single deletions were identified in both orientations. For (CTG•CAG)43 and (CTG•CAG)70, single, double and multiple deletions as well as inversions were detected (summarized in Supplementary Table 2). Moreover, for the former tract, single and double deletions were identified in both orientations and for the latter sequence single deletions were found in orientation I. In the other cases of the CTG•CAG sequence containing 43 and 70 repeats, mutagenic events were found only for orientation II. Additionally, a length dependence was also found because double deletions were observed between 0 and 20% of the cases for (CTG•CAG)43 and (CTG•CAG)70 in orientation II, respectively. Similar correlations were observed for inversions where 57.1 and 60% of the cases were detected in orientation II for CTG•CAG harboring 43 and 70 repeats, respectively. The orientation effect may be explained by the presence of CTG•CAG tracts attached to the GFP gene in orientation II (Figure 1). Hence, the TRSs had a stronger mutagenic effect where the TRSs were in close proximity to the GFP reporter gene (Discussion). Three types of DSB repair products were found (Discussion); first, plasmids where the entire TRS was deleted, second, plasmids with shortened TRSs where the EcoRI recognition site inside the tract was deleted and third, plasmids where the TRS was shortened but the EcoRI recognition site was preserved (Figure 4).


Double-strand breaks in the myotonic dystrophy type 1 and the fragile X syndrome triplet repeat sequences induce different types of mutations in DNA flanking sequences in Escherichia coli.

Kosmider B, Wells RD - Nucleic Acids Res. (2006)

Influence of the type and orientation of TRS on mutagenic patterns of DSB repair. The percentages of DSB repair products involving single, double and multiple deletions as well as inversions identified in the mutant clones are shown for plasmids harboring: (A) (CGG·CCG)43, in (B) (CTG·CAG)43 and in (C) (CTG·CAG)70. The orientations of the TRSs were explained in the legend to Figure 1. White bars indicate orientation I, whereas filled bars show orientation II. The very small percentages near zero are actually zero but are shown as larger values on this Figure to enable visualization. The data for this Figure was derived from Figure 2. Percentages are shown for each type of product for a given orientation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636463&req=5

fig3: Influence of the type and orientation of TRS on mutagenic patterns of DSB repair. The percentages of DSB repair products involving single, double and multiple deletions as well as inversions identified in the mutant clones are shown for plasmids harboring: (A) (CGG·CCG)43, in (B) (CTG·CAG)43 and in (C) (CTG·CAG)70. The orientations of the TRSs were explained in the legend to Figure 1. White bars indicate orientation I, whereas filled bars show orientation II. The very small percentages near zero are actually zero but are shown as larger values on this Figure to enable visualization. The data for this Figure was derived from Figure 2. Percentages are shown for each type of product for a given orientation.
Mentions: In summary, we found an influence of the type, orientation and the length of the repeat tracts on the induction of the mutagenic events (Figure 3). For (CGG•CCG)43, only single deletions were identified in both orientations. For (CTG•CAG)43 and (CTG•CAG)70, single, double and multiple deletions as well as inversions were detected (summarized in Supplementary Table 2). Moreover, for the former tract, single and double deletions were identified in both orientations and for the latter sequence single deletions were found in orientation I. In the other cases of the CTG•CAG sequence containing 43 and 70 repeats, mutagenic events were found only for orientation II. Additionally, a length dependence was also found because double deletions were observed between 0 and 20% of the cases for (CTG•CAG)43 and (CTG•CAG)70 in orientation II, respectively. Similar correlations were observed for inversions where 57.1 and 60% of the cases were detected in orientation II for CTG•CAG harboring 43 and 70 repeats, respectively. The orientation effect may be explained by the presence of CTG•CAG tracts attached to the GFP gene in orientation II (Figure 1). Hence, the TRSs had a stronger mutagenic effect where the TRSs were in close proximity to the GFP reporter gene (Discussion). Three types of DSB repair products were found (Discussion); first, plasmids where the entire TRS was deleted, second, plasmids with shortened TRSs where the EcoRI recognition site inside the tract was deleted and third, plasmids where the TRS was shortened but the EcoRI recognition site was preserved (Figure 4).

Bottom Line: DSBs at TRS junctions with the vector generated a large number of mutagenic events in flanking sequences whereas DSBs within the repeats elicited no similar products.Surprisingly, DNA sequence analyses on mutant clones revealed the presence of only single deletions of 0.4-1.6 kb including the TRS and the flanking sequence from plasmids originally containing (CGG*CCG)43 but single, double and multiple deletions as well as insertions were found for plasmids originally containing (CTG*CAG)n (where n = 43 or 70).Non-B DNA structures (slipped structures with loops, cruciforms, triplexes and tetraplexes) as well as microhomologies are postulated to participate in the recombination and/or repair processes.

View Article: PubMed Central - PubMed

Affiliation: Center for Genome Research, Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Texas Medical Center, 2121 W. Holcombe Boulevard, Houston, TX 77030-3303, USA.

ABSTRACT
The putative role of double-strand breaks (DSBs) created in vitro by restriction enzyme cleavage in or near CGG*CCG or CTG*CAG repeat tracts on their genetic instabilities, both within the repeats and in their flanking sequences, was investigated in an Escherichia coli plasmid system. DSBs at TRS junctions with the vector generated a large number of mutagenic events in flanking sequences whereas DSBs within the repeats elicited no similar products. A substantial enhancement in the number of mutants was caused by transcription of the repeats and by the absence of recombination functions (recA-, recBC-). Surprisingly, DNA sequence analyses on mutant clones revealed the presence of only single deletions of 0.4-1.6 kb including the TRS and the flanking sequence from plasmids originally containing (CGG*CCG)43 but single, double and multiple deletions as well as insertions were found for plasmids originally containing (CTG*CAG)n (where n = 43 or 70). Non-B DNA structures (slipped structures with loops, cruciforms, triplexes and tetraplexes) as well as microhomologies are postulated to participate in the recombination and/or repair processes.

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Related in: MedlinePlus