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Restriction endonuclease TseI cleaves A:A and T:T mismatches in CAG and CTG repeats.

Ma L, Chen K, Clarke DJ, Nortcliffe CP, Wilson GG, Edwardson JM, Morton AJ, Jones AC, Dryden DT - Nucleic Acids Res. (2013)

Bottom Line: The cleavage of targets containing these mismatches is as efficient as cleavage of the correct target sequence containing a central A:T base pair.The cleavage mechanism does not apparently use a base flipping mechanism as found for some other type II restriction endonuclease recognizing similarly degenerate target sequences.The ability of TseI to cleave targets with mismatches means that it can cleave the unusual DNA hairpin structures containing A:A or T:T mismatches formed by the repetitive DNA sequences associated with Huntington's disease (CAG repeats) and myotonic dystrophy type 1 (CTG repeats).

View Article: PubMed Central - PubMed

Affiliation: EaStChem School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh EH9 3JJ, UK.

ABSTRACT
The type II restriction endonuclease TseI recognizes the DNA target sequence 5'-G^CWGC-3' (where W = A or T) and cleaves after the first G to produce fragments with three-base 5'-overhangs. We have determined that it is a dimeric protein capable of cleaving not only its target sequence but also one containing A:A or T:T mismatches at the central base pair in the target sequence. The cleavage of targets containing these mismatches is as efficient as cleavage of the correct target sequence containing a central A:T base pair. The cleavage mechanism does not apparently use a base flipping mechanism as found for some other type II restriction endonuclease recognizing similarly degenerate target sequences. The ability of TseI to cleave targets with mismatches means that it can cleave the unusual DNA hairpin structures containing A:A or T:T mismatches formed by the repetitive DNA sequences associated with Huntington's disease (CAG repeats) and myotonic dystrophy type 1 (CTG repeats).

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Polyacrylamide gel analysis of matched and mismatched DNA duplexes being cut by TseI. Lane M was the molecular mass marker with 10, 15, 20 and 30 bp DNA indicated. DNA cleavage of A:T, T:T and A:A duplexes by TseI yields four 12–16mer single-strand oligonucleotides matching those shown in the products lane.
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gkt176-F3: Polyacrylamide gel analysis of matched and mismatched DNA duplexes being cut by TseI. Lane M was the molecular mass marker with 10, 15, 20 and 30 bp DNA indicated. DNA cleavage of A:T, T:T and A:A duplexes by TseI yields four 12–16mer single-strand oligonucleotides matching those shown in the products lane.

Mentions: Rather than perform full enzyme activity studies on each possible mismatch using the fluorescence assay, we performed gel assays, Figure 3, and HPLC assays, Supplementary Figure S5, for cleavage on duplexes containing various base pairs or mismatches in the central position of the target sequence. Figure 3 shows that 28-bp duplexes containing mismatches of A or T were all cleaved into shorter duplexes as effectively as the normal cognate sequence, but that those duplexes containing G or C at the central position were not cleaved as expected, as they lacked the target sequence. Cleavage was complete for the A:T , T:T and A:A duplexes as would be predicted from the kinetic parameters determined in the continuous fluorescence assay.Figure 3.


Restriction endonuclease TseI cleaves A:A and T:T mismatches in CAG and CTG repeats.

Ma L, Chen K, Clarke DJ, Nortcliffe CP, Wilson GG, Edwardson JM, Morton AJ, Jones AC, Dryden DT - Nucleic Acids Res. (2013)

Polyacrylamide gel analysis of matched and mismatched DNA duplexes being cut by TseI. Lane M was the molecular mass marker with 10, 15, 20 and 30 bp DNA indicated. DNA cleavage of A:T, T:T and A:A duplexes by TseI yields four 12–16mer single-strand oligonucleotides matching those shown in the products lane.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643589&req=5

gkt176-F3: Polyacrylamide gel analysis of matched and mismatched DNA duplexes being cut by TseI. Lane M was the molecular mass marker with 10, 15, 20 and 30 bp DNA indicated. DNA cleavage of A:T, T:T and A:A duplexes by TseI yields four 12–16mer single-strand oligonucleotides matching those shown in the products lane.
Mentions: Rather than perform full enzyme activity studies on each possible mismatch using the fluorescence assay, we performed gel assays, Figure 3, and HPLC assays, Supplementary Figure S5, for cleavage on duplexes containing various base pairs or mismatches in the central position of the target sequence. Figure 3 shows that 28-bp duplexes containing mismatches of A or T were all cleaved into shorter duplexes as effectively as the normal cognate sequence, but that those duplexes containing G or C at the central position were not cleaved as expected, as they lacked the target sequence. Cleavage was complete for the A:T , T:T and A:A duplexes as would be predicted from the kinetic parameters determined in the continuous fluorescence assay.Figure 3.

Bottom Line: The cleavage of targets containing these mismatches is as efficient as cleavage of the correct target sequence containing a central A:T base pair.The cleavage mechanism does not apparently use a base flipping mechanism as found for some other type II restriction endonuclease recognizing similarly degenerate target sequences.The ability of TseI to cleave targets with mismatches means that it can cleave the unusual DNA hairpin structures containing A:A or T:T mismatches formed by the repetitive DNA sequences associated with Huntington's disease (CAG repeats) and myotonic dystrophy type 1 (CTG repeats).

View Article: PubMed Central - PubMed

Affiliation: EaStChem School of Chemistry, University of Edinburgh, The King's Buildings, Edinburgh EH9 3JJ, UK.

ABSTRACT
The type II restriction endonuclease TseI recognizes the DNA target sequence 5'-G^CWGC-3' (where W = A or T) and cleaves after the first G to produce fragments with three-base 5'-overhangs. We have determined that it is a dimeric protein capable of cleaving not only its target sequence but also one containing A:A or T:T mismatches at the central base pair in the target sequence. The cleavage of targets containing these mismatches is as efficient as cleavage of the correct target sequence containing a central A:T base pair. The cleavage mechanism does not apparently use a base flipping mechanism as found for some other type II restriction endonuclease recognizing similarly degenerate target sequences. The ability of TseI to cleave targets with mismatches means that it can cleave the unusual DNA hairpin structures containing A:A or T:T mismatches formed by the repetitive DNA sequences associated with Huntington's disease (CAG repeats) and myotonic dystrophy type 1 (CTG repeats).

Show MeSH
Related in: MedlinePlus