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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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Model of mutagenic events triggered by DSBs introduced by EcoRV at CGG·CCG or CTG·CAG repeats. The filled and gray boxes represent the TRS with interruptions (EcoRI recognition site) and the GFP gene, respectively. The ends of the TRS created by DSBs may form alternative DNA conformations which may be processed by structure and/or repeat sequence specific proteins and/or nucleases. The homologous nucleotides between the breakpoints may serve as substrates for DSB repair which leads to the deletion events. All sequenced mutant clones had no functional reporter GFP gene (white CFUs) and contained: in (A), no TRS because of the deletion of the entire repeat tracts with a part of GFP gene; in (B), the shortened TRS where both the interruption (EcoRI recognition site), a part of the TRS and the GFP gene was deleted and, in (C), a portion of the TRS with the interruption but the EcoRI site was retained.
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fig4: Model of mutagenic events triggered by DSBs introduced by EcoRV at CGG·CCG or CTG·CAG repeats. The filled and gray boxes represent the TRS with interruptions (EcoRI recognition site) and the GFP gene, respectively. The ends of the TRS created by DSBs may form alternative DNA conformations which may be processed by structure and/or repeat sequence specific proteins and/or nucleases. The homologous nucleotides between the breakpoints may serve as substrates for DSB repair which leads to the deletion events. All sequenced mutant clones had no functional reporter GFP gene (white CFUs) and contained: in (A), no TRS because of the deletion of the entire repeat tracts with a part of GFP gene; in (B), the shortened TRS where both the interruption (EcoRI recognition site), a part of the TRS and the GFP gene was deleted and, in (C), a portion of the TRS with the interruption but the EcoRI site was retained.

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)

Model of mutagenic events triggered by DSBs introduced by EcoRV at CGG·CCG or CTG·CAG repeats. The filled and gray boxes represent the TRS with interruptions (EcoRI recognition site) and the GFP gene, respectively. The ends of the TRS created by DSBs may form alternative DNA conformations which may be processed by structure and/or repeat sequence specific proteins and/or nucleases. The homologous nucleotides between the breakpoints may serve as substrates for DSB repair which leads to the deletion events. All sequenced mutant clones had no functional reporter GFP gene (white CFUs) and contained: in (A), no TRS because of the deletion of the entire repeat tracts with a part of GFP gene; in (B), the shortened TRS where both the interruption (EcoRI recognition site), a part of the TRS and the GFP gene was deleted and, in (C), a portion of the TRS with the interruption but the EcoRI site was retained.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636463&req=5

fig4: Model of mutagenic events triggered by DSBs introduced by EcoRV at CGG·CCG or CTG·CAG repeats. The filled and gray boxes represent the TRS with interruptions (EcoRI recognition site) and the GFP gene, respectively. The ends of the TRS created by DSBs may form alternative DNA conformations which may be processed by structure and/or repeat sequence specific proteins and/or nucleases. The homologous nucleotides between the breakpoints may serve as substrates for DSB repair which leads to the deletion events. All sequenced mutant clones had no functional reporter GFP gene (white CFUs) and contained: in (A), no TRS because of the deletion of the entire repeat tracts with a part of GFP gene; in (B), the shortened TRS where both the interruption (EcoRI recognition site), a part of the TRS and the GFP gene was deleted and, in (C), a portion of the TRS with the interruption but the EcoRI site was retained.
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.

Show MeSH
Related in: MedlinePlus