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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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Diagram of plasmids and scheme of study. All plasmids contain interrupted CGG·CCG or CTG·CAG repeats with EcoRV and EcoRI recognition sites (in bold) neighboring and inside the TRS, respectively. The inserts (25) were cloned into pGFPTΔE harboring a transcription terminator cassette (13). The control pGFPTΔE contains no TRS. In orientation I, the (CGG)n or (CTG)n strand is the template for leading strand synthesis; in orientation II, the (CGG)n or (CTG)n strand is the template for lagging strand synthesis (25,40). Vertically cross-hatched arrow, ampicillin resistance gene (AmpR); horizontally cross-hatched arrow, unidirectional pUC19 origin of replication (Ori); dotted box, transcription terminator cassette (Ter); open arrow, lacZ promoter-operator (Pr); solid gray bar, LacZ–GFP fusion gene; solid black bar, triplet repeats. The CGG·CCG or CTG·CAG tracts were cloned into pGFPTΔE vector. DSBs were introduced by digestion of these plasmids with restriction enzymes. Linearized plasmids were immediately transformed into E.coli and the transformation mixtures were spread on LB agar plates. Individual white CFUs were analyzed using biochemical analyses and the sequences of the mutant clones were determined to evaluate if the breakpoints were flanked by non-B DNA conformations.
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fig1: Diagram of plasmids and scheme of study. All plasmids contain interrupted CGG·CCG or CTG·CAG repeats with EcoRV and EcoRI recognition sites (in bold) neighboring and inside the TRS, respectively. The inserts (25) were cloned into pGFPTΔE harboring a transcription terminator cassette (13). The control pGFPTΔE contains no TRS. In orientation I, the (CGG)n or (CTG)n strand is the template for leading strand synthesis; in orientation II, the (CGG)n or (CTG)n strand is the template for lagging strand synthesis (25,40). Vertically cross-hatched arrow, ampicillin resistance gene (AmpR); horizontally cross-hatched arrow, unidirectional pUC19 origin of replication (Ori); dotted box, transcription terminator cassette (Ter); open arrow, lacZ promoter-operator (Pr); solid gray bar, LacZ–GFP fusion gene; solid black bar, triplet repeats. The CGG·CCG or CTG·CAG tracts were cloned into pGFPTΔE vector. DSBs were introduced by digestion of these plasmids with restriction enzymes. Linearized plasmids were immediately transformed into E.coli and the transformation mixtures were spread on LB agar plates. Individual white CFUs were analyzed using biochemical analyses and the sequences of the mutant clones were determined to evaluate if the breakpoints were flanked by non-B DNA conformations.

Mentions: pRW5503 and pRW5504 containing CGG•CCG and pRW5511, pRW5512, pRW5509 and pRW5510 harboring CTG•CAG repeats (Figure 1) were prepared and characterized as follows. To obtain inserts in orientation I, where the (CGG)n or (CTG)n strands are the templates for leading strand synthesis, the TRS containing fragments from pRW4531, pRW4525 and pRW4523 (25) were released by Acc65I and HincII digestions. These inserts had, in addition to the 43 CGG•CCG repeats in pRW5503, the 43 CTG•CAG repeats in pRW5511 and the 70 CTG•CAG repeats in pRW5509, 13 bp of non-repetitive flanking sequences at the Acc65I site and 15 bp at the HincII site. The pGFPTΔE vector was digested with BsiWI and StuI because Acc65I and BsiWI have compatible sticky ends and HincII and StuI have blunt ends. On the other hand, for the plasmid preparations with the inserts in orientation II, where the (CGG)n or (CTG)n strands are the templates for lagging strand synthesis, the TRS containing fragments from pRW4531, pRW4525 and pRW4523 (25) were released by XbaI and SmaI digestions. In addition to the 43 CGG•CCG repeats in pRW5504, the 43 CTG•CAG repeats in pRW5512 and the 70 CTG•CAG repeats in pRW5510, these inserts had 7 bp of non-repetitive flanking sequences at the XbaI site and 12 bp at the SmaI site. These fragments were individually ligated to the pGFPTΔE vector digested with SpeI and StuI since XbaI and SpeI have compatible sticky ends and SmaI and StuI have blunt ends.


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)

Diagram of plasmids and scheme of study. All plasmids contain interrupted CGG·CCG or CTG·CAG repeats with EcoRV and EcoRI recognition sites (in bold) neighboring and inside the TRS, respectively. The inserts (25) were cloned into pGFPTΔE harboring a transcription terminator cassette (13). The control pGFPTΔE contains no TRS. In orientation I, the (CGG)n or (CTG)n strand is the template for leading strand synthesis; in orientation II, the (CGG)n or (CTG)n strand is the template for lagging strand synthesis (25,40). Vertically cross-hatched arrow, ampicillin resistance gene (AmpR); horizontally cross-hatched arrow, unidirectional pUC19 origin of replication (Ori); dotted box, transcription terminator cassette (Ter); open arrow, lacZ promoter-operator (Pr); solid gray bar, LacZ–GFP fusion gene; solid black bar, triplet repeats. The CGG·CCG or CTG·CAG tracts were cloned into pGFPTΔE vector. DSBs were introduced by digestion of these plasmids with restriction enzymes. Linearized plasmids were immediately transformed into E.coli and the transformation mixtures were spread on LB agar plates. Individual white CFUs were analyzed using biochemical analyses and the sequences of the mutant clones were determined to evaluate if the breakpoints were flanked by non-B DNA conformations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1636463&req=5

fig1: Diagram of plasmids and scheme of study. All plasmids contain interrupted CGG·CCG or CTG·CAG repeats with EcoRV and EcoRI recognition sites (in bold) neighboring and inside the TRS, respectively. The inserts (25) were cloned into pGFPTΔE harboring a transcription terminator cassette (13). The control pGFPTΔE contains no TRS. In orientation I, the (CGG)n or (CTG)n strand is the template for leading strand synthesis; in orientation II, the (CGG)n or (CTG)n strand is the template for lagging strand synthesis (25,40). Vertically cross-hatched arrow, ampicillin resistance gene (AmpR); horizontally cross-hatched arrow, unidirectional pUC19 origin of replication (Ori); dotted box, transcription terminator cassette (Ter); open arrow, lacZ promoter-operator (Pr); solid gray bar, LacZ–GFP fusion gene; solid black bar, triplet repeats. The CGG·CCG or CTG·CAG tracts were cloned into pGFPTΔE vector. DSBs were introduced by digestion of these plasmids with restriction enzymes. Linearized plasmids were immediately transformed into E.coli and the transformation mixtures were spread on LB agar plates. Individual white CFUs were analyzed using biochemical analyses and the sequences of the mutant clones were determined to evaluate if the breakpoints were flanked by non-B DNA conformations.
Mentions: pRW5503 and pRW5504 containing CGG•CCG and pRW5511, pRW5512, pRW5509 and pRW5510 harboring CTG•CAG repeats (Figure 1) were prepared and characterized as follows. To obtain inserts in orientation I, where the (CGG)n or (CTG)n strands are the templates for leading strand synthesis, the TRS containing fragments from pRW4531, pRW4525 and pRW4523 (25) were released by Acc65I and HincII digestions. These inserts had, in addition to the 43 CGG•CCG repeats in pRW5503, the 43 CTG•CAG repeats in pRW5511 and the 70 CTG•CAG repeats in pRW5509, 13 bp of non-repetitive flanking sequences at the Acc65I site and 15 bp at the HincII site. The pGFPTΔE vector was digested with BsiWI and StuI because Acc65I and BsiWI have compatible sticky ends and HincII and StuI have blunt ends. On the other hand, for the plasmid preparations with the inserts in orientation II, where the (CGG)n or (CTG)n strands are the templates for lagging strand synthesis, the TRS containing fragments from pRW4531, pRW4525 and pRW4523 (25) were released by XbaI and SmaI digestions. In addition to the 43 CGG•CCG repeats in pRW5504, the 43 CTG•CAG repeats in pRW5512 and the 70 CTG•CAG repeats in pRW5510, these inserts had 7 bp of non-repetitive flanking sequences at the XbaI site and 12 bp at the SmaI site. These fragments were individually ligated to the pGFPTΔE vector digested with SpeI and StuI since XbaI and SpeI have compatible sticky ends and SmaI and StuI have blunt ends.

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