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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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Fluorescence-based assay of TseI activity. (a) The 5′-HEX–labelled top strand was annealed with 3′-Black Hole Quencher (BHQ) 1–labelled strand and became highly quenched. Adding TseI at elevated temperature (60°C) results in cleavage of the duplex, separation of the fluorophore–quencher pair and the appearance of fluorescence. (b) Fluorescence intensity as a function of time. Initially, a low signal was observed. After 30 s, the sample chamber was opened, and an aliquot of TseI was added to 100 nM duplex (fluorescence assay A:T duplex, Table 1). After closing the sample chamber at 50 s, TseI caused a rapid increase in signal. (c) Time dependence of melting of the fluorescence assay product duplex at 60°C in the absence of any TseI. (d) Michaelis–Menten plots of TseI cleavage for both matched (open circles), A:A-mismatched DNA substrate (black circles) and T:T-mismatched DNA substrate (open square). Error bars are standard deviations for experiments performed in triplicate.
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gkt176-F2: Fluorescence-based assay of TseI activity. (a) The 5′-HEX–labelled top strand was annealed with 3′-Black Hole Quencher (BHQ) 1–labelled strand and became highly quenched. Adding TseI at elevated temperature (60°C) results in cleavage of the duplex, separation of the fluorophore–quencher pair and the appearance of fluorescence. (b) Fluorescence intensity as a function of time. Initially, a low signal was observed. After 30 s, the sample chamber was opened, and an aliquot of TseI was added to 100 nM duplex (fluorescence assay A:T duplex, Table 1). After closing the sample chamber at 50 s, TseI caused a rapid increase in signal. (c) Time dependence of melting of the fluorescence assay product duplex at 60°C in the absence of any TseI. (d) Michaelis–Menten plots of TseI cleavage for both matched (open circles), A:A-mismatched DNA substrate (black circles) and T:T-mismatched DNA substrate (open square). Error bars are standard deviations for experiments performed in triplicate.

Mentions: The endonuclease activity of TseI on short DNA duplexes containing the target sequence and variations thereof was investigated using a continuous fluorescence assay. The assay uses the difference in thermal stability of the 28 bp substrate and the shorter products, which will be single stranded at the assay temperature, to give a spectroscopic signal, Figure 2a. This assay was initially proposed by Waters et al. (23), who used the increase in absorption because of the melting of the short products, and was subsequently converted to fluorescence measurements by, for example, Li et al. (24), who used the melting of the short products to remove a fluorescence quencher from contact with a fluorescence HEX reporter. Provided that the assay temperature lies between the melting temperature of the substrate and the products, the products melt on cleavage by the enzyme, and the fluorescence of the fluorophore is greatly enhanced. This assay works well for TseI, Figure 2b, showing a substantial increase in fluorescence from a low-background level as a function of time after addition of the enzyme. The fluorescence assay product duplex melts faster than the cleavage of the substrate by the enzyme with melting complete after ∼60 s, Figure 2c. Thus the melting rate of the product does not limit the assay.Figure 2.


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)

Fluorescence-based assay of TseI activity. (a) The 5′-HEX–labelled top strand was annealed with 3′-Black Hole Quencher (BHQ) 1–labelled strand and became highly quenched. Adding TseI at elevated temperature (60°C) results in cleavage of the duplex, separation of the fluorophore–quencher pair and the appearance of fluorescence. (b) Fluorescence intensity as a function of time. Initially, a low signal was observed. After 30 s, the sample chamber was opened, and an aliquot of TseI was added to 100 nM duplex (fluorescence assay A:T duplex, Table 1). After closing the sample chamber at 50 s, TseI caused a rapid increase in signal. (c) Time dependence of melting of the fluorescence assay product duplex at 60°C in the absence of any TseI. (d) Michaelis–Menten plots of TseI cleavage for both matched (open circles), A:A-mismatched DNA substrate (black circles) and T:T-mismatched DNA substrate (open square). Error bars are standard deviations for experiments performed in triplicate.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt176-F2: Fluorescence-based assay of TseI activity. (a) The 5′-HEX–labelled top strand was annealed with 3′-Black Hole Quencher (BHQ) 1–labelled strand and became highly quenched. Adding TseI at elevated temperature (60°C) results in cleavage of the duplex, separation of the fluorophore–quencher pair and the appearance of fluorescence. (b) Fluorescence intensity as a function of time. Initially, a low signal was observed. After 30 s, the sample chamber was opened, and an aliquot of TseI was added to 100 nM duplex (fluorescence assay A:T duplex, Table 1). After closing the sample chamber at 50 s, TseI caused a rapid increase in signal. (c) Time dependence of melting of the fluorescence assay product duplex at 60°C in the absence of any TseI. (d) Michaelis–Menten plots of TseI cleavage for both matched (open circles), A:A-mismatched DNA substrate (black circles) and T:T-mismatched DNA substrate (open square). Error bars are standard deviations for experiments performed in triplicate.
Mentions: The endonuclease activity of TseI on short DNA duplexes containing the target sequence and variations thereof was investigated using a continuous fluorescence assay. The assay uses the difference in thermal stability of the 28 bp substrate and the shorter products, which will be single stranded at the assay temperature, to give a spectroscopic signal, Figure 2a. This assay was initially proposed by Waters et al. (23), who used the increase in absorption because of the melting of the short products, and was subsequently converted to fluorescence measurements by, for example, Li et al. (24), who used the melting of the short products to remove a fluorescence quencher from contact with a fluorescence HEX reporter. Provided that the assay temperature lies between the melting temperature of the substrate and the products, the products melt on cleavage by the enzyme, and the fluorescence of the fluorophore is greatly enhanced. This assay works well for TseI, Figure 2b, showing a substantial increase in fluorescence from a low-background level as a function of time after addition of the enzyme. The fluorescence assay product duplex melts faster than the cleavage of the substrate by the enzyme with melting complete after ∼60 s, Figure 2c. Thus the melting rate of the product does not limit the assay.Figure 2.

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